APC gene and nucleic acid probes derived therefrom

ABSTRACT

A human gene termed APC is disclosed. Methods and kits are provided for assessing mutations of the APC gene in hum tissues and body samples. APC mutations are found in familial adenomatous polyposis patients as wel as in sporadic colorectal cancer patents. APC is expressed in most normal tissue. These results suggest that APC is a tumor suppressor.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grantsawarded by the National Institutes of Health.

TECHNICAL AREA OF THE INVENTION

The invention relates to the area of cancer diagnostics andtherapeutics. More particularly, the invention relates to detection ofthe germline and somatie alterations of wild-type APC genes. Inaddition, it relates to therapeutic intervention to restore the functionof APC (adnomatous polyposis coli) gene product.

BACKGROUND OF THE INVENTION

According to the model of Knudson for tumorigenesis (Cancer Research,Vol. 45, p. 1482, 1985), there are tumor suppressor genes in all normalcells which, when they become non-functional due to mutation, causeneoplastic development. Evidence for this model has been found in theeases of retinoblastoma and colorectal tumors. The implicated suppressorgenes in those tumors, RB (retinoblastoma), p53 (protein having amolecular weight of 53 kDa), Dcc (deleted in colorectal cancer) and MCC(mutated in colorectal cancer) were found to be deleted or altered inmany eases of the tumors studied. (Hansen and Cavenee, Cancer Research,Vol.. 47, pp. 5518-5527 (1987): Baker et al., Science, Vol.. 244, p. 217(1989); Fearon et al., Science, Vol. 247, p. 49 (1990); Kinzler et al.Science Vol. 251. p. 1366 (1991).)

In order to fully understand the pathogenesis of tumors, it will benecessary to identify the other suppressor genes that play a role in thetumorigenesis process. Prominent among these is the one(s) presumptivelylocated at 5q21. Cytogenetie (Herrera et al., Am J. Med. Genet., Vol.25, p. 473 (1986) and linkage (Leppert et al., Science, Vol. 238, p.1411 (1987); Bodmer et al., Nature, Vol. 328, p. 614 (1987)) studieshave shown that this chromosome region harbors the gene responsible forfamilial adenomatous polyposis (FAP) and Gardner's Syndrome (GS). FAP isan autosomal-dominant, inherited disease in which affected individualsdevelop hundreds to thousands of adenomatous polyps, some of whichprogress to malignancy. GS is a variant of FAP in which desmoid tumors,osteomas and other soft tissue tumors occur together with multipleadenomas of the colon and rectum. A less severe form of polyposis hasbeen identified in which only a few (2-40) polyps develop. Thiscondition also is familial and is linked to the same chromosomal markersas FAP and GS (Leppert et al., New England Journal of Medicine, Vol.322, pp. 904-908, 1990.) Additionally, this chromosomal region is oftendeleted from the adenomas (Vogelstein et al., N. Engl. J. Med., Vol.319, p. 525 (1988)) and carcinomas (Vogelstein et al., N. Engl. J. Med.,Vol. 319, p. 525 (1988); Solomon et al., Nature, Vol. 328, p. 616(1987); Sasaki et al., Cancer Research. Vol. 49, p. 4402 (1989);Delattre et al., Lancet, Vol. 2, p. 353 (1989); and Ashton-Rickardt etal., Oncogene, Vol. 4, p. 1169 (1989)) of patients without FAP (sporadictumors). Thus, a putative suppressor gene on chromosome 5q21 appears toplay a role in the early stages of colorectal neoplasia in both sporadicand familial tumors.

Although the MCC gene has been identified on 5q21 as a candidatesuppressor gene, it does not appear to be altered in F AP or GSpatients. Thus there is a need in the art for investigations of thischromosomal region to identify genes and to determine if any of suchgenes are associated with FAP and/or GS and the process oftumorigenesis.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method fordiagnosing and prognosing a neoplastic tissue of a human.

It is another object of the invention to provide a method of detectinggenetic predisposition to cancer.

It is another object of the invention to provide a method of supplyingwild-type APC gene function to a cell which has lost said gene function.

It is yet another object of the invention to provide a kit fordetermination of the nucleotide sequence of APC alleles by thepolymerase chain reaction.

It is still another object of the invention to provide nucleic acidprobes for detection of mutations in the human APC gene.

It is still another object of the invention to provide a cDNA moleculeencoding the APC gene product.

It is yet another object or the invention to provide a preparation ofthe human APC protein.

It is another object of the invention to provide a method of screeningfor genetic predisposition to cancer.

It is an object of the invention to provide methods of testingtherapeutic agents for the ability to suppress neoplasia.

It is still another object of the invention to provide animals carryingmutant APC alleles.

These and other objects of the invention are provided by one or more ofthe embodiments which are described below. In one embodiment of thepresent invention a method of diagnosing or prognosing a neoplastictissue of a human is provided comprising: detecting somatic alterationof wild-type APC genes or their expression products in a sporadiccolorectal cancer tissue, said alteration indicating neoplasia of thetissue.

In yet another embodiment a method is provided of detecting geneticpredisposition to cancer in a human including familial adenomatouspolyposis (FAP) and Gardner's Syndrome (GS), comprising: isolating ahuman sample selected from the group consisting of blood and fetaltissue; detecting alteration of wild-type APC gene coding sequences ortheir expression products frown the sample, said alteration indicatinggenetic predisposition to cancer.

In another embodiment of the present invention a method is provided forsupplying wild-type APC gene function to a cell which has lost said genefunction by virtue of a mutation in the APC gene, comprising:introducing a wild-type APC gene into a cell which has lost said genefunction such that said wild-type gene is expressed in the cell.

In another embodiment a method of supplying wild-type APC gene functionto a cell is provided comprising: introducing a portion of a wild-typeAPC gene into a cell which has lost said gene function such that saidportion is expressed in the cell, said portion encoding a part of theAPC protein which is required for non-neoplastic growth of said cell.APC protein can also be applied to cells or administered to animals toremediate for mutant APC genes. Synthetic peptides or drugs can also beused to mimic APC function in cells which have altered APC expression.

In yet another embodiment a pair of single stranded primers is providedfor determination of the nucleotide sequence of the APC gene bypolymerase chain reaction. The sequence of said pair of single strandedDNA primers is derived from chromosome 5q band 21, said pair of primersallowing synthesis of APC gene coding sequences.

In still another embodiment of the invention a nucleic acid probe isprovided which is complementary to human wild-type APC gene codingsequences and which can form mismatches with mutant APC genes, therebyallowing their detection by enzymatic or chemical cleavage or by shiftsin electrophoretic mobility.

In another embodiment of the invention a method is provided fordetecting the presence of a neoplastic tissue in a human. The methodcomprises isolating a body sample from a human; detecting in said samplealteration of a wild-type APC gene sequence or wild-type APC expressionproduct, said alteration indicating the presence of a neoplastic tissuein the human.

In still another embodiment a cDNA molecule is provided which comprisesthe coding sequence of the APC gene.

In even another embodiment a preparation of the human APC protein isprovided which is substantially free of other human proteins. The aminoacid sequence of the protein is shown in .[.FIG. 3 or 7.]. .Iadd.FIGS.3A-3C or 7A-7W (SEQ ID NOS: 7 and 2).Iaddend..

In yet another embodiment of the invention a method is provided forscreening for genetic predisposition to cancer, including familialadenomatous polyposis (FAP) and Gardner's Syndrome (GS), in a human. Themethod comprises: detecting among kindred persons the presence of a DNApolymorphism which is linked to a mutant APC allele in an individualhaving a genetic predisposition to cancer, said kindred beinggenetically related to the individual, the presence of said polymorphismsuggesting a predisposition to cancer.

In another embodiment of the invention a method of testing therapeuticagents for the ability to suppress a neoplastically transformedphenotype is provided. The method comprises: applying a test substanceto a cultured epithelial cell which carries a mutation in an APC allele;and determining whether said test substance suppresses theneoplastically transformed phenotype of the cell.

In another embodiment of the invention a method of testing therapeuticagents for the ability to suppress a neoplastically transformedphenotype is provided. The method comprises: administering a testsubstance to an animal which carries a mutant APC allele; anddetermining whether said test substance prevents or suppresses thegrowth of tumors.

In still other embodiments of the invention transgenie animals areprovided. The animals carry a mutant APC allele from a second animalspecies or have been genetically engineered to contain an insertionmutation which disrupts an APC allele.

The present invention provides the art with the information that the APCgene, a heretofore unknown gene is, in fact, a target of mutationalalterations on chromosome 5q21 and that these alterations are associatedwith the process of tumorigenesis. This information allows highlyspecific assays to be performed to assess the neoplastic status of aparticular tissue or the predisposition to cancer of an individual. Thisinvention has applicability to Familial Adenomatous Polyposis, sporadiccolerectal cancers, Gardner's Syndrome, as well as the less severefamilial polyposis discusses above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an overview of yeast artificial chromosome (YAC) contigs(contiguous stretches of sequence). Genetic distances between selectedRFLP markers from within the contigs are shown in centiMorgans.

.[.FIG. 1B shows.]. .Iadd.FIGS. 1B-11, and -111 show .Iaddend.a detailedmap of the three central contigs. The position of the six identifiedgenes from within the FAP region is shown; the 5' and 3' ends of thetranscripts from these genes have in general not yet been isolated, asindicated by the string of dots surrounding the bars denoting the genespositions. Selected restriction endonuclease recognition sites areindicated. B, BssH2; S, SstII; M, MluI; N, NruI.

FIG. 2 shows the sequence of TB1 .Iadd.(SEQ ID NO:5) .Iaddend.and TB2.Iadd.(SEQ ID NO: 6) .Iaddend.genes. The cDNA sequence of the TB1 genewas determined from the analysis of 11 cDNA clones derived from normalcolon and liver, as described in the text. A total of 2314 bp werecontained within the overlapping cDNA clones, defining an ORF of 424amino acids beginning at nucleotide 1. Only the predicted amino acidsfrom the ORF are shown. The carboxy-terminal end of the ORF hasapparently been identified, but the 5' end of the TB1 transcript has notyet been precisely determined.

The cDNA sequence of the TB2 gene was determined from the YS-39 clonederived as described in the text. This clone consisted of 2300 bp anddefined an ORF of 185 amino acids beginning at nucleotide 1. Only thepredicted amino acids are shown. The carboxy terminal end of the ORF hasapparently been identified, but the 5' end of the TB2 transcript has notbeen precisely determined.

.[.FIG. 3 shows.]. .Iadd.FIGS. 3A-3C (collectively, FIG. 3) show.Iaddend.the sequence of the APC gene product .Iadd.(SEQ ID NO:7).Iaddend.. The cDNA sequence was determined through the analysis of 87cDNA clones derived from normal colon, liver, and brain. A total of 8973bp were contained within overlapping cDNA clones, defining an ORF of.[.2842.]. .Iadd.2843 .Iaddend.amino acids. In frame stop codonssurrounded this ORF, as described in the text, suggesting that theentire APC gene product was represented in the ORF illustrated. Only thepredicted amino acids are shown.

FIG. 4 shows the local similarity between human APC .Iadd.(SEQ ID NO: 2).Iaddend.and ral2 .Iadd.(SEQ ID NO: 8) .Iaddend.of yeast. Localsimilarity among the APC .Iadd.(SEQ ID NO: 2) .Iaddend.and MCC genes.Iadd.(SEQ ID NO: 10) .Iaddend.and the m3 muscarinic acetylcholinereceptor .Iadd.(SEQ ID NO: 9) .Iaddend.is shown. The region of the mAChRshown corresponds to that responsible for coupling the receptor to Gproteins. The connecting lines indicate identities; dots indicaterelated amino acids residues.

FIG. 5 shows the genomic map of the 1200 kb NotI fragment at the FAPlocus. The NotI fragment is shown as a bold line. Relevant parts of thedeletion chromosomes from patients 3214 and 3824 are shown as stippledlines. Probes used to characterize the NotI fragment and the deletions,and three YACs from which subclones were obtained, are shown below therestriction map. The chimeric end of YAC 183H12 is indicated by a dottedline. The orientation and approximate position of MCC are indicatedabove the map.

FIG. 6 shows the DNA sequence .Iadd.(SEQ ID NO: 3) .Iaddend.andpredicted amino acid sequence of DP1 (TB2) .Iadd.(SEQ ID NO:4).Iaddend.. The nucleotide numbering begins at the most 5' nucleotideisolated. A proposed initiation methionine (base 77) is indicated inbold type. The entire coding sequence is presented.

.[.FIG. 7 shows.]. .Iadd.FIGS. 7A-7W (collectively, FIG. 7) show.Iaddend.the cDNA .Iadd.(SEQ ID NO: 1) .Iaddend.and predicted amino acidsequence of DP2.5 (APC).[.,.]. .Iadd.(SEQ ID NO: 2). .Iaddend.Thenucleotide numbering begins at the proposed initiation methionine. Thenucleotides and amino acids of the alternatively spliced exon (exon 9;nucleotide positions .[.934-1236.]. .Iadd.957-1259.Iaddend.) arepresented in lower case letters. At the 3' end, a poly(A) additionsignal occurs at 9530, and one cDNA clone has a poly(A) at 9563. OthercDNA clones extend beyond 9563, however, and their consensus sequence isincluded here.

FIG. 8 shows the arrangement of exons in DP2.5 (APC).[., (A).]..Iadd.FIG. 8A. .Iaddend.Exon 9 corresponds to nucleotides 933-1312; exon9a corresponds m nucleotides 1236-1312. The stop codon in the cDNA is atnucleotide 8535. .[.(B).]. .Iadd.FIG. 8B .Iaddend.Partial intronicsequence surrounding each exon is shown .Iadd.(SEQ ID NOS: 11-38).5'intron sequences of exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,and 15 are shown in SEQ ID NOS: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, respectively, 3'intron sequences of exons 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, and 14 are shown in SEQ ID NOS: 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, respectively.Iaddend..

DETAILED DESCRIPTION

It is a discovery of the present invention that mutational eventsassociated with tumorigenesis occur in a previously unknown gene onchromosome 5q named here the APC (Adenomamus Polyposis Coli) gene.Although it was previously known that deletion of alleles on chromosome5q were common in certain types of cancers, it was not known that atarget gene of these deletions was the APC gene. Further it was notknown that other types of mutational events in the APC gene are alsoassociated with cancers, The mutations of the APC gene can involve grossrearrangements, such as insertions and deletions. Point mutations havealso been observed.

According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type APC gene is detected. "Alterationof a wild-type gene" according to the present invention encompasses allforms of mutations--including deletions. The alteration may be due toeither rearrangements such as insertions, inversions, and deletions, orto point mutations, Deletions may be of the entire gene or only aportion of the gene. Somatic mutations are those which occur only incertain tissues, e.g., in the tumor tissue, and are not inherited in thegermline. Germline mutations can be found in any of a body's tissues. Ifonly a single allele is somatieally mutated, an early neoplastic stateis indicated. However, if both alleles are mutated then a lateneoplastic state is indicated. The finding of APC mutations thusprovides both diagnostic and prognostic information. An APC allele whichis not deleted (e.g., that on the sister chromosome to a chromosomecarrying an APC deletion) can be screened for other mutations, such asinsertions, small deletions, and point mutations. It is believed thatmany mutations found in tumor tissues will be those leading to decreasedexpression of the APC gene product. However, mutations leading tonon-functional gene products would also lead to a cancerous state. Pointmutational events may occur in regulatory regions, such as in thepromoter of the gene, leading to loss or diminution of expression of themRNA. Point mutations may also abolish proper RNA processing, leading toloss of expression of the APC gene product.

In order to detect the alteration of the wild-type APC gene in a tissue,it is helpful to isolate the tissue free from surrounding normaltissues. Means for enriching a tissue preparation for tumor cells areknown in the art. For example, the tissue may be isolated from paraffinor cryostat sections. Cancer cells may also be separated from normalcells by flow cytometry. These as well as other techniques forseparating tumor from normal cells are well known in the art. If thetumor tissue is highly contaminated with normal cells detection ofmutations is more difficult.

Detection of point mutations may be accomplished by molecular cloning ofthe APC allele (or alleles) and sequencing that allele(s) usingtechniques well known in the art. Alternatively, the polymerase chainreaction (PCR) can be used to amplify gene sequences directly from agenomic DNA preparation from the tumor tissue. The DNA sequence of theamplified sequences can then be determined. The polymerase chainreaction itself is well known in the art. See, e.g., Saiki et al.,Science, Vol. 239, p. 487, 1988; U.S. Pat. No. 4,683,203; and U.S. Pat.No. 4,683,195. Specific primers which can be used in order to amplifythe gene will be discussed in more detail below. The ligase chainreaction, which is known in the art, can also be used to amplify APCsequences. See Wu et al., Genomics, Vol. 4, pp. 560-569 (1989). Inaddition, a technique known as allele specific PCR can be used. (SeeRuano and Kidd, Nucleic Acids Research, Vol. 17, p. 8392, 1989.)According to this technique, primers are used which hybridize at their3' ends to a particular APC mutation. If the particular APC mutation isnot present, an amplification product is not observed. AmplificationRefractory Mutation System (ALUMS) can also be used as disclosed inEuropean Patent Application Publication No. 0332435 and in Newton etal., Nucleic Acids Research, Vol. 17, p.7, 1989. Insertions anddeletions of genes can also be detected by cloning, sequencing andamplification. In addition, restriction fragment length polymorphism(RFLP) probes for the gene or surrounding marker genes can be used toscore alteration of an allele or an insertion in a polymorphic fragment.Such a method is particularly useful for screening among kindred personsof an affected individual for the presence of the APC mutation found inthat individual. Single stranded conformation polymorphism (SSCP)analysis can also be used to detect base change variants of an allele.(Orita et al., Proc. Natl. Acad. Sci. USA Vol. 86, pp. 2766-2770, 1989,and Genomics, Vol. 5, pp. 874-879, 1989.) Other techniques for detectinginsertions and deletions as are known in the art can be used.

Alteration of wild-type genes can also be detected on the basis of thealteration of a wild-type expression product of the gene. Suchexpression products include both the APC mRNA as well as the APC proteinproduct. The sequences of these products are shown in .[.FIGS. 3 and7.]. .Iadd.FIGS. 3A-3C and 7A-7W.Iaddend.. Point mutations may bedetected by amplifying and sequencing the mRNA or via molecular cloningof cDNA made from the mRNA. The sequence of the cloned cDNA can bedetermined using DNA sequencing techniques which are well known in theart. The cDNA can also be sequenced via the polymerase chain reaction(PCR) which will be discussed in more detail below.

Mismatches, according to the present invention are hybridized nucleicacid duplexes which are not 100% homologous. The lack of total homologymay be due to deletions, insertions, inversions, substitutions orframeshift mutations. Mismateh detection can be used to detect pointmutations in the gene or its mRNA product. While these techniques areless sensitive than sequencing, they are simpler to perform on a largenumber of tumor samples. An example of a mismateh cleavage technique isthe RNase protection method, which is described in detail in Winter etal., Proc. Natl. Acad. Sci. USA, Vol. 82, p. 7575, 1985 and Meyers etal., Science, Vol. 230, p. 1242, 1985. In the practice of the presentinvention the method involves the use of a labeled riboprobe which iscomplementary to the human wild-type AIsC gene coding sequence. Theriboprobe and either mRNA or DNA isolated from the tumor tissue areannealed (hybridized) together and subsequently digested with the enzymeRNase A which is able to detect some mismatches in a duplex RNAstructure. If a mismateh is detected by RNase A, it cleaves at the siteof the mismateh. Thus, when the annealed RNA preparation is separated onan electrophoretie gel matrix, if a mismateh has been detected andcleaved by RNase A, an RNA product will be seen which is smaller thanthe full-length duplex RNA for the riboprobe and the mRNA or DNA. Theriboprobe need not be the full length of the ArC mRNA or gene but can bea segment of either. If the riboprobe comprises only a segment of theArC mRNA or gene it will be desirable to use a number of these probes toscreen the whole mRNA sequence for mismatehes.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton et al., Proc. Natl.Acad. Sci. USA, Vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. Acad.Sci. USA, Vol. 72, p. 989, 1975. Alternatively, mismatches can bedetected by shifts in the electrophoretie mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, HumanGenetics, Vol. 42, p. 726, 1988. With either riboprobes or DNA probes,the cellular mRNA or DNA which might contain a mutation can be amplifiedusing PCR (see below) before hybridization. Changes in DNA of the ArCgene can also be detected using Southern hybridization, especially ifthe changes are gross rearrangements, such as deletions and insertions.

DNA sequences of the APC gene which have been amplified by use ofpolymerase chain reaction may also be screened using allele-specificprobes. These probes are nucleic acid oligomers, each of which containsa region of the APC gene sequence harboring a known mutation. Forexample, one oligomer may be about 30 nucleotides in length,corresponding to a portion of the APC gene sequence. By use of a batteryof such allele-specific probes, PCR amplification products can bescreened to identify the presence of a previously identified mutation inthe APC gene. Hybridization of allele-specific probes with amplified APCsequences can be performed, for example, on a nylon filter.Hybridization to a particular probe under stringent hybridizationconditions indicates the presence of the same mutation in the rumortissue as in the allele-specific probe.

Alteration of APC mRNA expression can be detected by any technique knownin the art. These include Northern blot analysis, PCR amplification andRNase protection. Diminished mRNA expression indicates an alteration ofthe wild-type APC gone.

Alteration of wild-type APC genes can also be detected by screening foralteration of wild-type APC protein. For example, monoclonal antibodiesimmunoreactive with APC can be used to screen a tissue. Lack of cognateantigen would indicate an APC mutation. Antibodies specific for productsof mutant alleles could also be used to detect mutant APC gene product.Such immunological assays can be done in any convenient format known inthe art. These include Western blots, immunohistochemical assays andELISA assays. Any means for detecting an altered APC protein can be usedto detect alteration of wild-type APC genes. Functional assays can beused, such as protein binding determinations. For example, it isbelieved that APC protein oligomerizes to itself and/or MCC protein orbinds to a G protein. Thus, an assay for the ability to bind to wildtype APC or MCC protein or that G protein can be employed. In addition,assays can be used which detect APC biochemical function. It is believedthat APC is involved in phospholipid metabolism. Thus, assaying theenzymatic products of the involved phospholipid metabolic pathway can beused to determine APC activity. Finding a mutant APC gene productindicates alteration of a wild-type APC gene.

Mutant APC genes or gene products can also be detected in other humanbody samples, such as, serum, stool, urine and sputum. The sametechniques discussed above for detection of mutant APC genes or geneproducts in tissues can be applied to other body samples. Cancer cellsare sloughed off from tumors and appear in such body samples. Inaddition, the APC gene product itself may be secreted into theextracellular space and found in these body samples even in the absenceof cancer cells. By screening such body samples, a simple earlydiagnosis can be achieved for many types of cancers. In addition, theprogress of chemotherapy or radiotherapy can be monitored more easily bytesting such body samples for mutant APC genes or gene products.

The methods of diagnosis of the present invention are applicable to anytumor in which APC has a role in tumorigenesis. Deletions of chromosomearm 5q have been observed in tumors of lung, breast, colon, rectum,bladder, liver, sarcomas, stomach and prostate, as well as in leukemiasand lymphomas. Thus these are likely to be tumors in which APC has arole. The diagnostic method of the present invention is useful forclinicians so that they can decide upon an appropriate course oftreatment. For example, a tumor displaying alteration of both APCalleles might suggest a more aggressive therapeutic regimen than a tumordisplaying alteration of only one APC allele.

The primer pairs of the present invention are useful for determinationof the nucleotide sequence of a particular APC allele using thepolymerase chain reaction. The pairs of single stranded DNA primers canbe annealed to sequences within or surrounding the APC gene onchromosome 5q in order to prime amplifying DNA synthesis of the APC geneitself. A complete set of these primers allows synthesis of all of thenucleotides of the APC gene coding sequences, i.e., the exons. The setof primers preferably allows synthesis of both intron and exonsequences. Allele specific primers can also be used. Such primers annealonly to particular APC mutant alleles, and thus will only amplify aproduct in the presence of the mutant allele as a template.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme site sequences appended to their 5'ends. Thus, all nucleotides of the primers are derived from APCsequences or sequences adjacent to APC except the few nucleotidesnecessary to form a restriction enzyme site. Such enzymes and sites arewell known in the art. The primers themselves can be synthesized usingtechniques which are well known in the art. Generally, the primers canbe made using oligonueleotide synthesizing machines which arecommercially available. Given the sequence of the APC open reading frameshown in .[.FIG. 7.]. .Iadd.FIGS. 7A-7W (SEQ ID NO: 1).Iaddend., designof particular primers is well within the skill of the art.

The nueleic acid probes provided by the present invention are useful fora number of purposes. They can be used in Southern hybridization togenomie DNA and in the RNase protection method for detecting pointmutations already discussed above. The probes can be used to detect PCRamplification products. They may also be used to detect mismatehes withthe APC gene or mRNA using other techniques. Mismatehes can be detectedusing either enzymes (e.g., S1 nuclease), chemicals (e.g., hydroxylamineor osmium tetroxide and piperidine), or changes in electrophoretiemobility of mismatched hybrids as compared to totally matched hybrids.These techniques are known in the art. See, Cotton, supra, Shenk, supra,Myers, supra, Winter, supra, and Novack et al., Proc. Natl. Acad. Sci.USA, Vol. 83, p. 586, 1986. Generally, the probes are complementary toAPC gene coding sequences, although probes to certain introns are alsocontemplated. An entire battery of nueleic acid probes is used tocompose a kit for detecting alteration of wild-type APC genes. The kitallows for hybridization to the entire APC gene. The probes may overlapwith each other or be contiguous.

If a riboprobe is used to detect mismatehes with mRNA, it iscomplementary to the mRNA of the human wild-type APC gene. The riboprobethus is an anti-sense probe in that it does not code for the APC proteinbecause it is of the opposite polarity to the sense strand. Theriboprobe generally will be labeled with a radioactive, colorimetic, orfluorometric material, which can be accomplished by any means known inthe art. If the riboprobe is used to detect mismatches with DNA it canbe of either polarity, sense or anti-sense. Similarly, DNA probes alsomay be used to detect mismatches.

Nucleic acid probes may also be complementary to mutant alleles of theAPC gene. These are useful to detect similar mutations in other patientson the basis of hybridization rather than mismatehes. These arediscussed above and referred to as allele-specific probes. As mentionedabove, the APC probes can also be used in Southern hybridizations togenomic DNA to detect gross chromosomal changes such as deletions andinsertions. The probes can also be used to select cDNA clones of APCgenes from tumor and normal tissues. In addition, the probes can be usedto detect APC mRNA in tissues to determine if expression is diminishedas a result of alteration of wild-type APC genes. Provided with the APCcoding sequence shown in .[.FIG. 7.]. .Iadd.FIGS. 7A-7W .Iaddend.(SEQ IDNO:1), design of particular probes is well within the skill of theordinary artisan.

According to the present invention a method is also provided ofsupplying wild-type APC function to a cell which carries mutant APCalleles. Supplying such function should suppress neoplastic growth ofthe recipient cells. The wild-type APC gene or a part of the gene may beintroduced into the cell in a vector such that the gene remainsextrachromosomal. In such a situation the gene will be expressed by thecell from the extrachromosomal location. If a gene portion is introducedand expressed in a cell carrying a mutant APC allele, the gene portionshould encode a part of the APC protein which is required fornon-neoplastic growth of the cell. More preferred is the situation wherethe wild-type APC gene or a part of it is introduced into the mutantcell in such a way that it recombines with the endogenous mutant APCgene present in the cell. Such recombination requires a doublerecombination event which results in the correction of the APC genemutation. Vectors for introduction of genes both for recombination andfor extrachromosomal maintenance are known in the art and any suitablevector may be used. Methods for introducing DNA into cells such aselectropotation, calcium phosphate co-precipitation and viraltransduction are known in the art and the choice of method is within thecompetence of the routineer. Cells transformed with the wild-type APCgene can be used as model systems to study cancer remission and drugtreatments which promote such remission.

Similarly, cells and animals which carry a mutant APC allele can be usedas model systems to study and test for substances which have potentialas therapeutic agents. The cells are typically cultured epithelialcells. These may be isolated from individuals with APC mutations, eithersomatic or germline. Alternatively, the cell line can be engineered tocarry the mutation in the APC allele. After a test substance is appliedto the cells, the neoplastically transformed phenotype of the cell willbe determined. Any trait of neoplastically transformed cells can beassessed, including anchorage-independent growth, tumorigenicity in nudemice, invasiveness of cells, and growth factor dependence. Assays foreach of these traits are known in the art.

Animals for testing therapeutic agents can be selected after mutagenesisof whole animals or after treatment of germline cells or zygotes. Suchtreatments include insertion of mutant APC alleles, usually from asecond animal species, as well as insertion of disrupted homologousgenes. Alternatively, the endogenous APC gene(s) of the animals may bedisrupted by insertion or deletion mutation. After test substances havebeen administered to the animals, the growth of tumors must be assessed.If the test substance prevents or suppresses the growth of tumors, thenthe test substance is a candidate therapeutic agent for the treatment ofFAP and/or sporadic cancers.

Polypeptides which have APC activity can be supplied to cells whichcarry mutant or missing APC alleles. The sequence of the APC protein isdisclosed in .[.FIG. 3 or 7 (SEQ ID NO:7 or 1).]. .Iadd.FIGS. 3A-3C and7A-7W (SEQ ID NOS: 2 or 7).Iaddend.. .[.These two sequences differslightly and appear to be indicate the existence of two different formsof the APC protein..]. Protein can be produced by expression of the cDNAsequence in bacteria, for example, using known expression vectors.Alternatively, APC can be extracted from APC-producing mammalian cellssuch as brain cells. In addition, the techniques of synthetic chemistrycan be employed to synthesize APC protein. Any of such techniques canprovide the preparation of the present invention which comprises the APCprotein. The preparation is substantially free of other human proteins.This is most readily accomplished by synthesis in a microorganism or invitro.

Active APC molecules can be introduced into cells by microinjection orby use of liposomes, for example. Alternatively, some such activemolecules may be taken up by cells, actively or by diffusion.Extracellular application of APC gene product may be sufficient toaffect tumor growth. Supply of molecules with APC activity should leadto a partial reversal of the neoplastic state. Other molecules with APCactivity may also be used to effect such a reversal, for examplepeptides, drugs, or organic compounds.

The present invention also provides a preparation of antibodiesimmunoreactive with a human APC protein. The antibodies may bepolyclonal or monoclonal and may be raised against native APC protein,APC fusion proteins, or mutant APC proteins. The antibodies should beimmunoreactive with APC epitopes, preferably epitopes not present onother human proteins. In a preferred embodiment of the invention theantibodies will immunoprecipitate APC proteins from solution as well asreact with APC protein on Western or immunoblots of polyacrylamide gels.In another preferred embodiment, the antibodies will detect APC proteinsin paraffin or frozen tissue sections, using immunocytochemicaltechniques. Techniques for raising and purifying antibodies are wellknown in the art and any such techniques may be chosen to achieve thepreparation of the invention.

Predisposition to cancers as in FAP and GS can be ascertained by testingany tissue of a human for mutations of the APC gene. For example, aperson who has inherited a germline APC mutation would be prone todevelop cancers. This can be determined by testing DNA from any tissueof the person's body. Most simply, blood can be drawn and DNA extractedfrom the cells of the blood. In addition, prenatal diagnosis can beaccomplished by testing fetal cells, placental cells, or amniotic fluidfor mutations of the APC gene. Alteration of a wild-type APC allele,whether for example, by point mutation or by deletion, can be detectedby any of the means discussed above.

Molecules of cDNA according to the present invention are intron-free,APC gene coding molecules. They can be made by reverse transcriptaseusing the APC mRNA-as a template. These molecules can be propagated invectors and cell lines as is known in the art. Such molecules have thesequence shown in SEQ ID NO: 7. The cDNA can also be made using thetechniques of synthetic chemistry given the sequence disclosed herein.

A short region of homology has been identified between APC and the humanm3 muscarinic acetylcholine receptor (mAChR). This chornology waslargely confined to 29 residues in which 6 out of 7 amino acids(EL(GorA)GLQA) were identical (See FIG. 4 .Iadd.(SEQ ID NO:9).Iaddend.). Initially, it was not known whether this hornology wassignificant, because many other proteins had higher levels of globalhornology (though few had six out of seven contiguous amino acids incommon). However, a study on the sequence elements controlling G proteinactivation by mAChR subtypes (Lechleiter et al., EMBO J., p. 4381(1990)) has shown that a 21 amino acid region from the m3 mAChRcompletely mediated G protein specificity when substituted for the 21amino acids of m2 mAChR at the analogous protein position. These 21residues overlap the 19 amino acid hornology between APC and m3 mAChR.

This connection between APC and the G protein activating region of mAChRis intriguing in light of previous investigations relating G proteins tocancer. For example, the RAS oneogenes, which are often mutated incolorectal cancers (Vogelstein, et al., N. Engl. J. Med., Vol. 319, p.525 (1988); Bos et al., Nature Vol. 327, p. 293 (1987)), are members ofthe G protein family (Bourne, et al., Nature, Vol. 348, p. 125 (1990))as is an in vitro transformation suppressor (Noda et al., Proc. Natl.Acad. Sci. USA, Vol. 86, p. 162 (1989)) and genes mutated in hormoneproducing tumors (Candis et al., Nature, Vol. 340, p. 692 (1989); Lyonset al., Science, Vol. 249, p. 655 (1990)). Additionally, the generesponsible for neurofibromatosis (presumably a tumor suppressor gene)has been shown to activate the GTPase activity of RAS (Xu et al., Cell,Vol. 63, p. 835 (1990); Martin et al., Cell, Vol. 63, p. 843 (1990);Ballester et al., Cell, Vol. 63, p. 851 (1990)). Another interestinglink between G proteins and colon cancer involves the drug sulindae.This agent has been shown to inhibit the growth of benign colon tumorsin patients with FAP, presumably by virtue of its activity as acyclooxygenase inhibitor (Waddell et al., J. Surg. Oncology 24(1), 83(1983); Wadell, et al., Am. J. Surg., 157(1), 175 (1989); Charneau etal., Gastroenterologie Clinique at Biologique 14(2), 153 (1990)).Cyclooxygenase is required to convert arachidonic acid to prostaglandinsand other biologically active molecules. G proteins are known toregulate phospholipase A2 activity, which generates arachidonic acidfrom phospholipids (Role et al., Proc. Natl. Acad. Sci. USA, Vol. 84, p.3623 (1987); Kurachi et al., Nature, Vol. 337, 12 555 (1989)). Thereforewe propose that wild-type APC protein functions by interacting with a Gprotein and is involved in phospholipid metabolism.

The following are provided for exemplification purposes only and are notintended to limit the scope of the invention which has been described inbroad terms above.

EXAMPLE 1

This example demonstrates the isolation of a 5.5 Mb region of human DNAlinked to the FAP locus. Six genes are identified in this region, all ofwhich are expressed in normal colon cells and in colorectal, lung, andbladder tumors.

The cosmid markers YN5.64 and YN5.48 have previously been shown todelimit an 8 cM region containing the locus for FAP (Nakamura et al.,Am. J. Hum. Genet. Vol. 43, p. 638 (1988)). Further linkage andpulse-field gel electrophoresis (PFGE) analysis with additional markershas shown that the FAP locus is contained within a 4 cM region borderedby cosmids EF5.44 and L5.99. In order to isolate clones representing asignificant portion of this locus, a yeast artificial chromosome (YAC)library was screened with various 5q21 markers. Twenty-one YAC clones,distributed within six contigs and including 5.5 Mb from the regionbetween YN5.64 and YN5.48, were obtained (FIG. 1A).

Three contigs encompassing approximately 4 Mb were contained within thecentral portion of this region. The YAC's constituting these contigs,together with the markers used for their isolation and orientations, areshown in FIG. 1. These YAC contigs were obtained in the following way.To initiate each contig, the sequence of a genomic marker cloned fromchromosome 5q21 was determined and used to design primers for PCR. PCRwas then carried out on pools of YAC clones distributed in mierotitertrays as previously described (Anand et al., Nucleic Acids Research,Vol. 18, p. 1951 (1980)). Individual YAC clones from the positive poolswere identified by further PCR or hybridization based assays, and theYAC sizes were determined by PFGE.

To extend the areas covered by the original YAC clones, "chromosomalwalking" was performed. For this purpose, YAC termini were isolated by aPCR based method and sequenced (Riley et al., Nucleie Acids Research,Vol. 18, p. 2887 (1990)). PCR primers based on these sequences were thenused to rescreen the YAC library. For example, the sequence from anintron of the FER gene (Hao et al., Mol. Cell. Biol., Vol. 9, p. 1587(1989)) was used to design PCR primers for isolation of the 28EC1 and5EH8 YACs. The termini of the 28EC1 YAC were sequenced to derive markersRHE28 and LHE28, respectively. The sequences of these two markers werethen used to isolate YAC clones 15CH12 (from RHE28) and 40CF1 and 29EF1(from LHE28). These five YAC's formed a config encompassing 1200 kb(contig 1, FIG. 1B).

Similarly, contig 2 was initiated using cosmid N5.66 sequences, andcontig 3 was initiated using sequences both from the MCC gene and fromcosmid EF5.44. A walk in the telomeric direction from YAC 14FH1 and awalk in the opposite direction from YAC 39GG3 allowed connection of theinitial contig 3 clones through YAC 37HG4 (FIG. 1B). .Iadd.YAC37HG4 wasdeposited at the National Collection of Industrial and Marine Bacteria(NCIMB), P.O. Box 31, 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland,under Accession No. 4035A, FB3 on Dec. 17, 1990. .Iaddend.

Multipoint linkage analysis with the various markers used to define thecontigs, combined with PFGE analysis, showed that contigs 1 and 2 werecentromecic to contig 3. These contigs were used as tools to orientand/or identify genes which might be responsible for FAP. Six genes werefound to lie within this cluster of YAC's, as follows:

Contig #1: FER - The FER gene was discovered through its hornology tothe vital oncogene ABL (Hao et al., supra). It has an intrinsic tyrosinekinase activity, and in situ hybridization with an FER probe showed thatthe gene was located at 5q11-23 (Morris et al., Cytogenet. Cell. Genet.,Vol. 53, p. 4, (1990)). Because of the potential role of thisoncogene-related gene in neoplasia, we decided to evaluate it furtherwith regards to the FAP locus. A human genomic clone from FER wasisolated (MF 2.3) and used to define a restriction fragment lengthpolymorphism (RFLP), and the RFLP in turn used to map FER by linkageanalysis using a panel of three generation families. This showed thatFER was very tightly linked to previously defined polymorphic markersfor the FAP locus. The genetic mapping of FER was complemented byphysical mapping using the YAC clones derived from FER sequences (FIG.1B). Analysis of YAC contig 1 showed that FER was within 600 kb ofcosmid marker M5.28, which maps to within 1.5 Mb of cosmid L5.99 by PFGEof human gertomit DNA. Thus, the YAC mapping results were consistentwith the FER linkage data and PFGE analyses.

Contig 2:TB1 - TB1 was identified through a cross-hybridizationapproach. Exons of genes are often evolutionarily conserved whileintrons and intergenie regions are much less conserved. Thus, if a humanprobe cross-hybridizes strongly to the DNA from non-primate species,there is a reasonable chance that it contains exon sequences. Subclonesof the cosraids shown in FIG. 1 were used to .[.semen.]. .Iadd.screen.Iaddend.Southern blots containing rodent DNA samples. A subclone ofcosmid N5.66 (p 5.66-4) was shown to strongly hybridize to rodent DNA,and this clone was used to .[.semen.]. .Iadd.screen .Iaddend.cDNAlibraries derived from normal adult colon and fetal liver. The ends ofthe initial eDNA clones obtained in this screen were then used to extendthe eDNA sequence. Eventually, 11 cDNA clones were isolated, covering2314 bp. The gene detected by these clones was named TB1. Sequenceanalysis of the overlapping clones revealed an open reading frame (ORF)that extended for 1302 bp starting from the most 5' sequence dataobtained (FIG. 2A). If this entire open reading frame were translated,it would encode 434 amino acids .Iadd.(SEQ ID NO: 5).Iaddend.. Theproduct of this gene was not globally homologous to any other sequencein the current database but showed two significant local similarities toa family of ADP, ATP carrier/translocator proteins and mitochondrialbrown fat uncoupling proteins which are widely distributed from yeast tomammals. These conserved regions of TB1 (underlined in FIG. 2A) maydefine a predictive motif for this sequence family. In addition, TB1appeared to contain a signal peptide (or mitochondrial targetingsequence) as well as at least 7 transmembrane domains.

Conrig 3: MCC, TB2, SRP and APC - The MCC gene was also discoveredthrough a cross-hybridization approach, as described previously (Kinzleret al., Science Vol. 251, p. 1366 (1991)). The MCC gene was considered acandidate for causing FAP by virtue of its tight genetic linkage to FAPsusceptibility and its somatic mutation in sporadic colorectalcarcinomas. However, mapping experiments suggested that the codingregion of MCC was approximately 50 kb proximal to the centromeric end ofa 200 kb deletion found in an FAP patient. MCC cDNA probes detected a 10kb mRNA transcript on Northern blot analysis of which 4151 bp, includingthe entire open reading frame, have been cloned. Although the 3'non-translated portion or an alternatively spliced form of MCC mighthave extended into this deletion, it was possible that the deletion didnot affect the MCC gene product. We therefore used MCC sequences toinitiate a YAC contig, and subsequently used the YAC clones to identifygenes 50 to 250 kb distal to MCC that might be contained within thedeletion.

In a first approach, the insert from YAC24ED6 (FIG. 1B) wasradiolabelled and hybridized to a cDNA library from normal colon. One ofthe cDNA clones (YS39) identified in this manner detected a 3.1 kb mRNAtranscript when used as a probe for Northern blot hybridization.Sequence analysis of the YS39 clone revealed that it encompassed 2283nucleotides and contained an ORF that extended for 555 bp from the most5' sequence data obtained. If all of this ORF were translated, it wouldencode 185 amino acids .Iadd.(SEQ ID NO: 6) .Iaddend.(FIG. 2B). The genedetected by YS39 was named TB2. Searches of nucleotide and proteindatabases revealed that the TB2 gene was not identical to any previouslyreported sequences nor were there any striking similarities.

Another clone (YS11) identified through the YAC 24ED6 screen appeared tocontain portions of two distinct genes. Sequences from one end of YS11were identical to at least 180 bp of the signal recognition particleprotein SRP19 (Lingelbach et al. Nucleic Acids Research, Vol. 16, p.9431 (1988). A second ORF, from the opposite end of clone YS11, provedto be identical to 78 bp of a novel gene which was independentlyidentified through a second YAC-based approach. For the latter, DNA fromyeast cells containing YAC 14FH1 (FIG. 1B) was digested with EcoRI andsubcloned into a plasmid vector. Plasmids that contained human DNAfragments were selected by colony hybridization using total human DNA asa probe. These clones were then used to search for cross-hybridizingsequences as described above for TB1, and the cross-hybridizing cloneswere subsequently used to screen cDNA libraries. One of the cDNA clonesdiscovered in this way (FH38) contained a long ORF (2496 bp), 78 bp ofwhich were identical to the above-noted sequences in YS11. The ends ofthe FH38 cDNA clone were then used to initiate cDNA walking to extendthe sequence. Eventually, 85 cDNA clones were isolated from normalcolon, brain and liver cDNA libraries and found to encompass 8973nucleotides of contiguous transcript. The gene corresponding to thistranscript was named APC. When used as probes for Northern blotanalysis, APC cDNA clones hybridized to a single transcript ofapproximately 9.5 kb, suggesting that the great majority of the geneproduct was represented in the cDNA clones obtained. Sequences from the5' end of the APC gene were found in YAC 37HG4 but not in YAC 14FH1.However, the 3' end of the APC gene was found in 14FH1 as well as 37HG4.Analogously, the 5' end of the MCC coding region was found in YAC clones19AA9 and 26GC3 but not 24ED6 or 14FH1, while the 3' end displayed theopposite pattern. Thus, MCC and APC transcription units pointed inopposite directions, with the direction of transcription going fromcentromeric to telomeric in the case of MCC, and telomeric tocentromeric in the case of APC. PFGE analysis of YAC DNA digested withvarious restriction endonucleases showed that TB2 and SRP were betweenMCC and APC, and that the 3' ends of the coding regions of MCC and APCwere separated by approximately 150 kb (FIG. 1B).

Sequence analysis of the APC cDNA clones revealed an open reading frameof 8,535 nucleotides. The 5' end of the ORF contained a methionine codon(codon 1) that was preceded by an in-frame stop codon 9 bp upstream, andthe 3' end was followed by several in-frame stop codons. The proteinproduced by initiation at codon 1 would contain .[.2,842.]. .Iadd.2843.Iaddend.amino acids .[.(FIG. 3).]. .Iadd.FIGS. 3A-3C (SEQ ID NO:7).Iaddend.. The results of database searching with the APC gene productwere quite complex due to the presence of large segments with locallybiased amino acid compositions. In spite of this, APC could be roughlydivided into two domains. The N-terminal 25% of the protein had a highcontent of leueine residues (12%) and showed local sequence similaritiesto myosins, various intermediate filament proteins (e.g., desrain,vimentin, neurofilaments) and Drosophila armadillo/human plakoglobin.The latter protein is a component of adhesive junctions (desmosomes)joining epithelial cells (Franke et al., Proc. Natl. Acad. Sci. U.S.A.,Vol. 86, p. 4027 (1989); Perlet et al., Cell, Vol. 63, p. 1167 (1990))The C-terminal 75% of APC (residues 731- 2832) is 17% serine bycomposition with setinc residues more or less uniformly distributed.This large domain also contains local concentrations of charged (mostlyacidic) and proline residues. There was no indication of potentialsignal peptides, transmembrane regions, or nuclear targeting signals inAPC, suggesting a cytoplasmic localization.

To detect short similarities to APC, a database search was performedusing the PAM-40 matrix (Altsehul. J. Mol. Bio., Vol. 219, p. 555(1991). Potentially interesting matches to several proteins were found.The most suggestive of these involved the ral2 gene product of yeast,which is implicated in the regulation of ras activity (Fukul et al.,Mol. Cell. Biol., Vol. 9, p. 5617 (1989)). Little is known about howral2 might interact with ras but it is interesting to note thepositively-charged character of this region in the context of thenegatively-charged GAP interaction region of ras. A specificelectrostatic interaction between ras and GAP-related proteins has beenproposed.

Because of the proximity of the MCC and APC genes, and the fact thatboth are implicated in colorectal tumorigenesis, we searched forsimilarities between the two predicted proteins. Bourne has previouslynoted that MCC has the potential to form alpha helical coiled coils(Nature, Vol. 351, p. 188 (1991). Lupas and colleagues have recentlydeveloped a program for predicting coiled coil potential from primarysequence data (Science, Vol. 252, p. 1162 (1991) and we have used theirprogram to analyze both MCC and APC. Analysis of MCC indicated adiscontinuous pattern of coiled-coil domains separated by putative"hinge" or "spacer" regions similar to those seen in laminin and otherintermediate filament proteins. Analysis of the APC sequence revealedtwo regions in the N-terminal domain which had strong coiledcoil-forming potential, and these regions corresponded to those thatshowed local similarities with myosin and IF proteins on databasesearching. In addition, one other putative coiled coil region wasidentified in the central region of APC. The potential for both APC andMCC to form coiled coils is interesting in that such structures oftenmediate homo- and hetero-oligomerization.

Finally, it had previously been noted that MCC shared a short similaritywith the region of the m3 muscarinic acetylcholine receptor (mAChR)known to regulate specificity of G-protein coupling. The APC gene alsocontained a local similarity to the region of the m3 mAChR .Iadd.(SEQ IDNO: 9) .Iaddend.that overlapped with the MCC similarity .Iadd.(SEQ IDNO: 10) .Iaddend.(FIG. 4B). Although the similarities to ral2 .Iadd.(SEQID NO: 8) .Iaddend.(FIG. 4A) and m3 mAChR .Iadd.(SEQ ID NO: 9).Iaddend.(FIG. 4B) were not statistically significant, they wereintriguing in light of previous observations relating G-proteins toneoplasia.

Each of the six genes described above was expressed in normal colonmucosa, as indicated by their representation in colon cDNA libraries. Tostudy expression of the genes in neoplastic colorectal epithelium, weemployed reverse transcription-polymerase chain reaction (PCR) assays.Primers based on the sequences of FER, TB1, TB2, MCC, and APC were eachused to design primers for PCR performed with cDNA templates. Each ofthese genes was found to be expressed in normal colon, in each of tencell lines derived from colorectal cancers, and in tumor cell linesderived from lung and bladder tumors. The ten colorectal cancer celllines included eight from patients with sporadic CRC and two frompatients with FAP.

EXAMPLE 2

This example demonstrates a genetic analysis of the role of the FER genein FAP and sporadic colorectal cancers.

We considered FER as a candidate because of its proximity to the FAPlocus as judged by physical and genetic criteria (see Example 1), andits homology to known tyrosine kinases with oncogenic potential. Primerswere designed to PCR-amplify the complete coding sequence of FER fromthe RNA of two colorectal cancer cell lines derived from FAP patients.cDNA was generated from RNA and used as a template for PCR. The primersused were 5'-.[.AGAAGGATCCCTTGTGCAGTGTGGA.]..Iadd.AGAAGGATCCCTTGTGCAGTGTGGA.Iaddend.-3'.Iadd.(SEQ ID NO: 95).Iaddend.and 5'-GACAGGATCCTGAAGCTGAGTTTG-3'.Iadd.(SEQ ID NO:96).Iaddend.. The underlined nucleotides were altered from the true FERsequence to create BamHI sites. The cell lines used were JW and Dill,both derived from colorectal cancers of FAP patients. (C. Paraskeva, B.G. Buckle, D. Sheer, C. B. Wigley, Int. J. Cancer 34, 49 (1984); M. E.Gross et al., Cancer Res. 51, 1452 (1991). The resultant 2554 basepairfragments were cloned and sequenced in their entirety. The PCR productswere cloned in the BamHI site of Bluescript SK (Stratagene) and pools ofat least 50 clones were sequenced en masse using T7 polymerase, asdescribed in Nigro et al., Nature 342,705 (1989).

Only a single conservative amino acid change (GTG-<CTG, creating a valto leu substitution at codon 439) was observed. The region surroundingthis codon was then amplified from the DNA of individuals without FAPand this substitution was found to be a common polymorphism, notspecifically associated with FAP. Based on these results, we consideredit unlikely (though still possible) the FER gene was responsible forFAP. To amplify the regions surrounding codon 439, the following primerswere used: 5'-TCAGAAAGTGCTGAAGAG-3' .Iadd.(SEQ ID NO: 97) .Iaddend.and5'-GGAATAATTAGGTCTCCAA-3' .Iadd.(SEQ ID NO: 98).Iaddend.. PCR productswere digested with PstI, which yields a 50 bp fragment if codon 439 isleucine, but 26 and 24 bp fragments if it is valine. The primers usedfor sequencing were chosen from the FER cDNA sequence in Hao et al.,supra.

EXAMPLE 3

This example demonstrates the genetic analysis of MCC, TB2, SRP and APCin FAP and sporadic rolorectal tumors. Each of these genes is linked andencompassed by conrig 3 (see FIG. 1).

Several lines of evidence suggested that this conrig was of particularinterest. First, at least three of the four genes in this conrig werewithin the deleted region identified in two FAP patients. (See Example 5infra.) Second, allelic deletions of chromosome 5q21 in sporadic cancersappeared to be centered in this region. (Ashton-Rickardt et al.,Oncogene, in press; and Miki et al., Japn. J. Cancer Res., in press.)Some tumors exhibited loss of proximal RFLP markers (up to andpotentially including the 5' end of MCC), but no loss of markers distalto MCC. Other tumors exhibited loss of markers distal to and perhapsincluding the 3' end of MCC, but no loss of sequences proximal to MCC.This suggested either that different ends of MCC were affected by lossin all such cases, or alternatively, that two genes (one proximal to andperhaps including MCC, the other distal to MCC) were separate targets ofdeletion. Third, clones from each of the six FAP region genes were usedas probes on Southern blots containing tumor DNA from patients withsporadic CRC. Only two examples of somatic changes were observed in over200 tumors studied: a rearrangement/deletion whose centromeric end waslocated within the MCC gene (Kinzler et al., supra) and an 800 bpinsertion within the APC gene between nucleotides 4424 and 5584. Fourth,point mutations of MCC were observed in two tumors (Kinzler et al.)supra strongly suggesting that MCC was a target of mutation in at leastsome sporadic colorectal cancers.

Based on these results, we attempted to search for subtle alterations ofconrig 3 genes in patients with FAP. We chose to examine MCC and APC,rather than TB2 or SRP, because of the somatic mutations in MCC and APCnoted above. To facilitate the identification of subtle alterations, thegenomic sequences of MCC and APC exons were determined (see TableI.Iadd.; SEQ ID NOS: 24-38).Iaddend.. These sequences were used todesign primers for PCR analysis of constitutional DNA from FAP patients.

We first amplified eight exons and surrounding introns of the MCC genein affected individuals from 90 different FAP kindreds. The PCR productswere analyzed by a ribonuclease (RNase) protein assay. In brief, the PCRproducts were hybridized to in vitro transcribed RNA probes representingthe normal genomic sequences. The hybrids were digested with RNase A,which can cleave at single base pair mismatches within DNA-RNA hybrids,and the cleavage products were visualized following denaturing gelelectrophoresis. Two separate RNase protection analyses were performedfor each exon, one with the sense and one with the antisense strand.Under these conditions. approximately 40% of all mismatches aredetectable. Although some amino acid variants of MCC were observed inFAP patients, all such variants were found in a small percentage ofnormal individuals. These variants were thus unlikely to be responsiblefor the inheritance of FAP.

We next examined three exons of the APC gene. The three exons examinedincluded those containing nt 822-930, 931-1309, and the first 300 nt ofthe most distal exon (nt 1956-2256). PCR and RNase protection analysiswere performed as described in Kinzler et al. supra, using the primersunderlined in Table I .Iadd.(SEQ ID NO: 24-38).Iaddend.. The primers fornt 1956-2256 were 5'-GCAAATCCTAAGAGAGAACAA-3' .Iadd.(SEQ ID NO: 99).Iaddend.and 5'-GATGGCAAGCTTGAGCCAG-3'.Iadd.(SEQ ID NO: 100).Iaddend..

In 90 kindreds, the RNase protection method was used to screen formutations and in an additional 13 kindreds, the PCR products were clonedand sequenced to search for mutations not detectable by RNaseprotection. PCR products were cloned into a Bluescript vector modifiedas described in T. A. Holton and M. W. Graham, Nueleic Acids Res. 19,1156 (1991). A minimum of 100 clones were pooled and sequenced. Fivevariants were detected among the 103 kindreds analyzed. Cloning andsubsequent DNA sequencing of the PCR product of patient P21 indicated aC to T transition in codon 413 that resulted in a change from arginineto cysteine. This amino acid variant was not observed in any of 200 DNAsamples from individuals without FAP. Cloning and sequencing of the PCRproduct from patients P24 and P34, who demonstrated the same abnormalRNase protection pattern indicated that both had a C to T transition atcodon 301 that resulted in a change from arginine (CGA) to a stop codon(TGA). This change was not present in 200 individuals without FAP. Asthis point mutation resulted in the predicted loss of the recognitionsite for the enzyme Taq I, appropriate PCR products could be digestedwith Taq I to detect the mutation. This allowed us to determine that thestop codon co-segregated with disease phenotype in members of the familyof P24. The inheritance of this change in affected members of thepedigree provides additional evidence for the importance of themutation.

Cloning and sequencing of the PCR product from FAP patient P93 indicateda C to G transversion at codon 279, also resulting in a stop codon(change from TCA to TGA). This mutation was not present in 200individuals without FAP. Finally, one additional mutation resulting in aserine (TCA) to stop codon (TGA) at codon 712 was detected in a singlepatient with FAP (patient P60).

The five germline mutations identified are summarized in Table IIA, aswell as four others discussed in Example 9. In addition to thesegermline mutations, we identified several somatic mutations of MCC andAPC in sporadic CRC's. Seventeen MCC exons were examined in 90 sporadiccolorectal cancers by RNase protection analysis. In each case where anabnormal RNase protection pattern was observed, the corresponding PCRproducts were cloned and sequenced. This led to the identification ofsix point mutations (two described previously) (Kinzler et al., supra),each of which was not found in the germline of these patients (TableIIB). Four of the mutations resulted in amino acid substitutions and tworesulted in the alteration of splice site consensus elements. Mutationsat analogous splice site positions in other genes have been shown toalter RNA processing in vivo and in vitro.

Three exons of APC were also evaluated in sporadic tumors. Sixty tumorswere screened by RNase protection, and an additional 98 tumors wereevaluated by sequencing. The exons examined included nt 822-930,931-1309, and 1406-1545 (Table I). A total of three mutations wereidentified, each of which proved to be somatic. Tumor T27 contained asomatic mutation of C GA (arginine) to TGA (stop codon) at codon 33.Tumor T135 contained a GT to GC change at a splice donor site. Tumor T34contained a 5 bp insertion (CAGCC between codons 288 and 289) resultingin a stop at codon 291 due to a frameshift.

We serendipitously discovered one additional somatic mutation in acolorectal cancer. During our attempt to define the sequences and splicepatterns of the MCC and APC gene products in colorectal epithelialcells, we cloned cDNA from the colorectal cancer cell line SW480. Theamino acid sequence of the MCC gene from SW480 was identical to thatpreviously found in clones from human brain. The sequence of APC inSW480 cells, however, differed significantly, in that a transition atcodon 1338 resulted in a change from glutamine (CAG) to a stop codon(TAG). To determine if this mutation was somatic, we recovered DNA fromarchival paraffin blocks of the original surgical specimen (T201) fromwhich the tumor cell line was derived 28 years ago.

DNA was purified from paraffin sections as described in S. E. Goelz, S.R. Hamilton, and B. Vogelstein. Bioehem. Biophys. Res. Comm. 130, 118(1985). PCR was performed using the primers 5'-GTTCCAGCAGTGTCACAG-3'.Iadd.(SEQ ID NO: 101) .Iaddend.and 5'-GGGAGATTTCGCTCCTGA-3' .Iadd.(SEQID NO: 102).Iaddend.. A PCR product containing codon 1338 was amplifiedfrom the archival DNA and used to show that the stop codon represented asomatic mutation present in the original primary tumor and in cell linesderived from the primary and metastatie tumor sites, but not from normaltissue of the patient.

The ten point mutations in the MCC and APC genes so far discovered insporadic CRCs are summarized in Table IIB. Analysis of the number ofmutant and wild-type PCR clones obtained from each of these tumorsshowed that in eight of the ten eases, the wild-type sequence waspresent in approximately equal proportions to the mutant. This wasconfirmed by RFLP analysis using flanking markers from chromosome 5qwhich demonstrated that only two of the ten tumors (T135 and T201)exhibited an allelie deletion on chromosome 5q. These results areconsistent with previous observations showing that 20-40% of sporadiccolorectal tumors had allelie deletions of chromosome 5q. Moreover,these data suggest that mutations of 5q21 genes are not limited to thosecolorectal tumors which contain allelic deletions of this chromosome.

EXAMPLE 4

This example characterizes small, nested deletions in DNA from twounrelated FAP patients.

DNA from 40 FAP patients was screened with cosmids that had been mappedinto a region near the APC locus to identify small deletions orrearrangements. Two of these cosmids, L5.71 and L5.79, hybridized with a1200 kb NotI fragment in DNAs from most or the FAP patients screened.

The DNA of one FAP patient, 3214, showed only a 940 kb NotI fragmentinstead of the expected 1200 kb fragment. DNA was analyzed from fourother members of the patient's immediate family; the 940 kb fragment waspresent in her affected mother (4711), but not in the other, unaffectedfamily members. The mother also carried a normal 1200 kb NotI fragmentthat was transmitted to her two unaffected offspring. These observationsindicated that the mutant polyposis allele is on the same chromosome asthe 940 kb NotI fragment. A simple interpretation is that APC patients3214 and 4711 each carry a 260 kb deletion within the APC locus.

If a deletion were present, then other enzymes might also expected toproduce fragments with altered mobilities. Hybridization of L5.79 toNruI-digested DNAs from both affected members of the family revealed anovel NruI fragment of 1300 kb, in addition to the normal 1200 kb NruIfragment. Furthermore, MluI fragments in patients 3214 and 4711 alsoshowed an increase in size consistent with the deletion of an MluI site.The two chromosome 5 homologs of patient 3214 were segregated in somaticcell hybrid lines; HHW1155 (deletion hybrid) carried the abnormalhomolog and HHW1159 (normal hybrid) carried the normal homolog.

Because patient 3214 showed only a 940 kb NotI fragment, she had notinherited the 1200 kb fragment present in the unaffected father's DNA.This observation suggests that he must be heterozygous for, and havetransmitted, either a deletion of the L5.79 probe region or a variantNotI fragment too large to resolve on the gel system. As expected, thehybrid cell line HHW1159, which carries the paternal homolog, revealedno resolved Not fragment when probed with L5.79. However, probing ofHHW1159 DNA with L5.79 following digestion with other enzymes did revealrestriction fragments, demonstrating the presence of DNA homologous tothe probe. The father is, therefore, interpreted as heterozygous for apolymorphism at the NotI site, with one chromosome 5 having a 1200 kbNotI fragment and the other having a fragment too large to resolveconsistently on the gel. The latter was transmitted to patient 3214.

When double digests were used to order restriction sites within the 1200kb NotI fragment, L5.71 and L5.79 were both found to lie on a 550 kbNotI-NruI fragment and, therefore, on the same side or an NruI site inthe 1200 kb NotI fragment. To obtain genomic representation of sequencespresent over the entire 1200 kb NotI fragment, we constructed a libraryof small-fragment inserts enriched for sequences from this fragment. DNAfrom the somatic cell hybrid HHW141, which contains about 40% ofchromosome 5, was digested with NotI and electrophoresed underpulsed-field gel (PFG) conditions; EcoRI fragments from the 1200 kbregion of this gel were cloned into a phage vector. Probe Map30 wasisolated from this library. In normal individuals probe Map30 hybridizesto the 1200 kb NotI fragment and to a 200 kb NruI fragment. This latterhybridization places Map30 disrat, with respect to the locations ofL5.71 and L5.79, to the NruI site of the 550 kb NotI-NruI fragment.

Because Map30 hybridized to the abnormal, 1300 kb NruI fragment ofpatient 3214, the locus defined by Map30 lies outside the hypothesizeddeletion. Furthermore, in normal chromosomes Map30 identified a 200 kbNruI fragment and L5.79 identified a 1200 kb NruI fragment; thehypothesized deletion must, therefore, be removing an NruI site, orsites, lying between Map30 and L5.79, and these two probes must flankthe hypothesized deletion. A restriction map of the genomic region,showing placement of these probes is shown in FIG. 5.

A NotI digest of DNA from another FAP patient, 3824, was probed withL5.79. In addition to the 1200 kb normal NotI fragment, a fragment ofapproximately 1100 kb was observed, consistent with the presence of a100 kb deletion in one chromosome 5. In this case, however, digestionwith NruI and MluI did not reveal abnormal bands, indicating that if adeletion were present, its boundaries must lie distal to the NruI andMluI sites of the fragments identified by L5.79. Consistent with thisexpectation, hybridization of Map30 to DNA from patient 3824 identifieda 760 kb MluI fragment in addition to the expected 860 kb fragment,supporting the interpretation of a 100 kb deletion in this patient. Thetwo chromosome 5 homologs of patient 3824 were segregated in somaticcell hybrid lines; HHW1291 was found to carry only the abnormal homologand HHW1290 only the normal homolog.

That the 860 kb MluI fragment identified by Map30 is distinct from the830 kb MluI fragment identified previously by L5.79 was demonstrated byhybridization of Map30 and L5.79 to a NotI-MluI double digest of DNAfrom the hybrid cell (HHW1159) containing the nondeleted chromosome 5homolog of patient 3214. As previously indicated, this hybrid isinterpreted as missing one of the NotI sites that define the 1200 kbfragment. A 620 kb NotI-MluI fragment was seen with probe L5.79, and an860 kb fragment was seen witch Map30. Therefore, the 830 kb MluIfragment recognized by probe L5.79 must contain a NotI site in HHW1159DNA; because the 860 kb MluI fragment remains intact, it does not carrythis NotI site and must be distinct from the 830 kb MluI fragment.

EXAMPLE 5

This example demonstrates the isolation of human sequences which spanthe region deleted in the two unrelated FAP patients characterized inExample 4.

A strong prediction of the hypothesis that patients 3214 and 3824 carrydeletions is that some sequences present on normal chromosome 5 homologswould be missing from the hypothesized deletion homologs. Therefore, todevelop gertomit probes that might confirm the deletions, as well as toidentify genes from the region, YAC clones from a conrig seeded bycosmid L5.79 were localized from a library containing seven haploidhuman genome equivalents (Albertsen et al., Proc. Natl. Acad. Sci.U.S.A., Vol. 87, pp. 4256-4260 (1990))with respect to the hypothesizeddeletions. Three clones, YACs 57B8, 310D8, and 183H12, were found tooverlap the deleted region.

Importantly, one end of YAC 57B8 (clone AT57) was found to lie withinthe patient 3214 deletion. Inverse polymerase chain reaction (PCR)defined the end sequences of the insert of YAC 57B8. PCR primers basedon one of these end sequences repeatedly failed to amplify DNA from thesomatic cell hybrid (HHW1155) carrying the deleted homolog of patient3214, but did amplify a product of the expected size from the somaticcell hybrid (HHW1159) carrying the normal chromosome 5 homolog. Thisresult supported the interpretation that the abnormal restrictionfragments found in the DNA of patient 3214 result from a deletion.

Additional support for the hypothesis of deletion in DNA from patient3214 came from subcloned fragments of YAC 183H12, which spans the regionin question. Y11, an EcoRI fragment cloned from YAC 183H12, hybridizedto the normal, 1200 kb NotI fragment of patient 4711, but failed tohybridize to the abnormal, 940 kb NotI fragment of 4711 or to DNA fromdeletion cell line HHW1155. This result confirmed the deletion inpatient 3214.

Two additional EcoR1 fragments from YAC 183H12, Y10 and Y14, werelocalized within the patient 3214 deletion by their failure hybridizieto DNA from HHW1155. Probe Y10 hybridizes to a 150 kb NruI fragment innormal chromosome 5 homologs. Because the 3214 deletion creates the 1300kb NruI fragment seen with the probes L5.79 and Map30 that flank thedeletion, these NruI sites and the 150 kb NruI fragment lying betweenmust be deleted in patient 3214. Furthermore, probe Y10 hybridizes tothe same 620 kb NotI-MluI fragment seen with probe L5.79 in normal DNA,indicating its location as L5.79-proximal to the deleted MluI site andplacing it between the Mlul site and the L5.79-proximal NruI site. TheMluI site must, therefore, lie between the NruI sites that define the150 kb NruI fragment (see FIG. 5).

Probe Y11 also hybridized to the 150 kb NruI fragment in the normalchromosome 5 homolog, but failed to hybridize to the 620 kb NotI-MluIfragment, placing it L5.79-distal to the MluI site, but proximal to thesecond NruI site. Hybridization to the same (860 kb) MluI fragment asMap30 confirmed the localization of probe Y11 L5.79-distal to the MluIsite.

Probe Y14 was shown to be L5.79-distal to both deleted NruI sites byvirtue of its hybridization to the same 200 kb NruI fragment of thenormal chromosome 5 seen with Map30. Therefore, the order of these EcoRIfragments derived from YAC 183H12 and deleted in patient 3214, withrespect to L5.79 and Map30, is L5.79-Y10-Y11-Y14-Map30.

The 100 kb deletion of patient 3824 was confirmed by the failure ofaberrant restriction fragments in this DNA to hybridize with probe Y11,combined with positive hybridizations to probes Y10 and/or Y14. Y10 andY14 each hybridized to the 1100 kb NotI fragment of patient 3824 as wellas to the normal 1200 kb NotI fragment, but Y11 hybridized to the 1200kb fragment only. In the MluI digest, probe Y14 hybridized to the 860 kband 760 kb fragments of patient 3824 DNA, but probe Y11 hybridized onlyto the 860 kb fragment. We conclude that the basis for the alteration infragment size in DNA from patient 3824 is, indeed, a deletion.Furthermore, because probes Y10 and Y14 are missing from the deleted3214 chromosome, but present on the deleted 3824 chromosome, and theyhave been shown to flank probe Y11, the deletion in patient 3824 must benested within the patient 3214 deletion.

Probes Y10, Y11, Y14 and Map30 each hybridized to YAC 310D8, indicatingthat this YAC spanned the patient 3824 deletion and at a minimum, mostof the 3214 deletion. The YAC characterizations. therefore, confirmedthe presence of deletions in the patients and provided physicalrepresentation of the deleted region.

EXAMPLE 6

This example demonstrates that the MCC coding sequence maps outside ofthe region deleted in the two FAP patients characterized in Example 4.

An intriguing FAP candidate gene, MCC, recently was ascertained withcosmid L5.71 and was shown to have undergone mutation in coloncarcinomas (Kinzler et al., supra). It was therefore of interest to mapthis gene with respect to the deletions in FAP patients. Hybridizationof MCC probes with an overlapping series of YAC clones extending ineither direction from L5.71 showed that the 3' end of MCC must beoriented toward the region of the two FAP deletions.

Therefore, two 3' cDNA clones from MCC were mapped with respect to thedeletions: clone 1CI (bp 2378-4181) and clone 7 (bp 2890-3560). Clone1CI contains sequences from the C-terminal end of the open readingframe, which stops at nucleotide 2708, as well as 3' untranslatedsequence. Clone 7 contains sequence that is entirely 3' to the openreading frame. Importantly, the entire 3' untranslated sequencecontained in the cDNA clones consists of a single 2.5 kb exon. These twoclones were hybridized to DNAs from the YACs spanning the FAP region.Clone 7 fails to hybridize to YAC 310D8, although it does hybridize toYACs 183H12 and 5738; the same result was obtained with the cDNA 1CI.Furthermore, these probes did show hybridization to DNAs from bothhybrid cell lines (HWW1159 and HWW1155) and the lymphoblastoid cell linefrom patient 3214, confirming their locations outside the deletedregion. Additional mapping experiments suggested that the 3' end of theMCC cDNA clone contig is likely to be located more than 45 kb from thedeletion of patient 3214 and, therefore, more than 100 kb from thedeletion of patient 3824.

EXAMPLE 7

This example identifies three genes within the deleted region ofchromosome 5 in the two unrelated FAP patients characterized in Example4.

Genomie clones were used to semen cDNA libraries in three separateexperiments. One screening was done with a phage clone derived from YAC310D8 known to span the 260 kb deletion of patient 3214. A large-insertphage library was constructed from this YAC; screening with Y11identified λ205, which mapped within both deletions. When clone λ205 wasused to probe a random-, plus oligo(dT)-, primed fetal brain cDNAlibrary (approximately 300,000 phage), six cDNA clones were isolated andeach of them mapped entirely within both deletions. Sequence analysis ofthese six clones formed a single cDNA contig, but did not reveal anextended open reading frame. One of the six cDNAs was used to isolatemore cDNA clones, some of which crossed the L5.71-proximal breakpoint ofthe 3824 deletion, as indicated by hybridization to both chromosome ofthis patient. These clones also contained an open reading frame,indicating a transcriptional orientation proximal to distal with respectto L5.71. This gene was named DP1 (deleted in polyposis 1). This gene isidentical to TB2 described above.

cDNA walks yielded a cDNA conrig of 3.0-3.5 kb, and included two clonescontaining terminal poly(A) sequences. This size corresponds to the 3.5kb band seen by Northern analysis. Sequencing of the first 3163 bp ofthe cDNA conrig revealed an open reading frame extending from the firstbase to nucleotide 631, followed by a 2.5 kb 3' untranslated region. Thesequence surrounding the methionine codon at base 77 conforms to theKozak consensus of an initiation methionine (Kozak, 1984). Failedattempts to walk farther, coupled with the similarity of the lengths ofisolated cDNA and mRNA, suggested that the NH₂ -terminus of the DP1protein had been reached. Hybridization to a combination of genomic andYAC DNAs cut with various enzymes indicated the genomic coverage of DP1to be approximately 30 kb.

Two additional probes for the locus, YS-11 and YS-39, which had beenascertained by screening of a cDNA library with an independent YAC probeidentified with MCC sequences adjacent to L5.71, were mapped into thedeletion region. YS-39 was shown to be a cDNA identical in sequence toDP1. Partial characterization of YS-11 had shown that 200 bp of DNAsequence at one end was identical to sequence coding for the 19 kdprotein of the ribosomal signal recognition particle. SRP19 (Lingelbachet al., supra). Hybridization experiments mapped YS-11 within bothdeletions. The sequence of this clone, however, was found to be complex.Although 454 bp of the 1032 bp sequence of YS-11 were identical to theGenBank entry for the SRP19 gene. another 578 bp appended 5' to theSRP19 sequence was found to consist of previously unreported sequencecontaining no extended open reading frames. This suggested that YS-11was either a chimetic clone containing two independent inserts or aclone of an incompletely processed or aberrant message. If YS-11 were aconventional chimetic clone, the independent segments would not beexpected to map to the same physical region. The segments resulting fromanomalous processing of a continuous transcript, however, would map to asingle chromosomai region.

Inverse PCR with primers specific to the two ends of YS-11, the SRP19end and the unidentified region, verified that both sequences map withinthe YAC 310D8; therefore, YS-11 is most likely a clone of an immature oranomalous mRNA species. Subsequently, both ends were shown to lie withthe deleted region of patient 3824, and YS-11 was used to screen foradditional cDNA clones.

Of the 14 cDNA clones selected from the fetal brain library, one clone,V5, was of particular interest in that it contained an open readingframe throughout, although it included only a short identity to thefirst 78 5' bases of the YS-11 sequence. Following the 78 bp ofidentical sequence, the two cDNA sequences diverged at an AG.Furthermore, divergence from genomie sequence was also seen after these78 bp, suggesting the presence of a splice junction, and supporting theview that YS-11 represents an irregular message.

Starting with V5, successive 5' and 3' walks were performed; theresulting cDNA contig consisted of more than 100 clones, which defined anew transcript, DP2. Clones walking in the 5' direction crossed the 3824deletion breakpoint farthest from L5.71; since its 3' end is closer tothis cosmid than its 5' end, the transcriptional orientation of DP2 isopposite to that of MCC and DP1.

The third screening approach relied on hybridization with a 120 kb MluIfragment from YAC 57B8. This fragment hybridizes with probe Y11 andcompletely spans the 100 kb deletion in patient 3824. the fragment waspurified on two preparative PFGs, labeled, and used to screen a fetalbrain cDNA library. A number of cDNA clones previously identified in thedevelopment of the DP1 and DP2 configs were reascertained. However, 19new cDNA clones mapped into the patient 3824 deletion. Analysisindicated that these 19 formed a new contig, DP3, containing a largeopen reading frame.

A clone from the 5' end of this new cDNA contig hybridized to the sameEcoRI fragment as the 3' end of DP2. Subsequently, the DP2 and DP3contigs were connected by a single 5' walking step from DP3, to form thesingle contig DP2.5. The complete nucleotide sequence of DP2.5 is shownin FIG. 7.

The consensus cDNA sequence of DP2.5 suggests that the entire codingsequence of DP2.5 has been obtained and is 8532 bp long. The most 5' ATGcodon occurs two codons from an in-frame stop and conforms to the Kozakinitiation consensus (Kozak, Nucl. Acids. Res., Vol. 12, p. 857-8721984). The 3' open reading frame breaks down over the final 1.8 kb,giving multiple stops in all frames. A poly(A) sequence was found in oneclone approximately 1 kb into the 3' untranslated region, associatedwith a polyadenylation signal 33 bp upstream (position 9530). The openreading frame is almost identical to that identified as APC above.

An alternatively spliced exon at nucleotide 934 of the DP2.5 transcriptis of potential interest. it was first discovered by noting that twoclasses of cDNA had been isolated. The more abundant cDNA class containsa 303 bp exon not included in the other. The presence in vivo of the twotranscripts was verified by an exon connection experiment. Primersflanking the alternatively spliced exon were used to amplify, by PCR,cDNA prepared from various adult tissues. Two PCR products that differedin size by approximately 300 bases were amplified from all the tissuestested; the larger product was always more abundant than the smaller.

EXAMPLE 8

This example demonstrates the primers used to identify subtle mutationsin DP1, SRP19, and DP2.5.

To obtain DNA sequence adjacent to the exons of the genes DP1, DP2.5,and SRP19, sequencing substrate was obtained by inverse PCRamplification of DNAs from two YACs, 310D8 and 183H12, that span thedeletions. Ligation at low concentration cyclized the restrictionenzyme-digested YAC DNAs. Oligonucleotides with sequencing tails,designed in inverse orientation at intervals along the cDNAs, primed PCRamplification from the cyclized templates. Comparison of these DNAsequences with the cDNA sequences placed exon boundaries at thedivergence points. SRP19 and DP1 were each shown to have five exons.DP2.5 consisted of 15 exons. The sequences of the oligonucleotidessynthesized to provide PCR amplification primers for the exons of eachof these genes are listed in Table III .Iadd.(SEQ ID NOS:39-94).Iaddend.. With the exception of exons 1, 3, 4, 9, and 15 of DP2.5(see below), the primer sequences were located in intron sequencesflanking the exons. The 5' primer of exon 1 is complementary to the cDNAsequence, but extends just into the 5' Kozak consensus sequence for theinitiator methionine, allowing a survey of the translated sequences. The5' primer of exon 3 is actually in the 5' coding sequences of this exon,as three separate intronic primers simply would not amplify. The 5'primer of exon 4 just overlaps the 5' end of this exon, and we thus failto survey the 19 most 5' bases of this exon. For exon 9, two overlappingprimer sets were used, such that each had one end within the exon. Forexon 15, the large 3' exon of DP2.5, overlapping primer pairs wereplaced along the length of the exon; each pair amplified a product of250-400 bases.

EXAMPLE 9

This example demonstrates the use of single stranded conformationpolymorphism (SSCP) analysis as described by Orita et al. Proc. Natl.Acad. Sci. U.S.A., Vol. 86, pp. 2766-70 (1989) and Genomies, Vol. 5, pp.874-879 (1989) as applied to DP1, SRP19 and DP2.5.

SSCP analysis identifies most single- or multiple-base changes in DNAfragments up to 400 bases in length. Sequence alterations are detectedas shifts in eleetrophoretie mobility of single-stranded DNA onnondenaturing aerylamide gels; the two complementary strands of a DNAsegment usually resolve as two SSCP conformers of distinct mobilities.However, if the sample is from an individual heterozygous for abase-pair variant within the amplified segment, often three or morebands are seen. In some eases, even the sample from a homozygousindividual will show multiple bands. Base-pair-change variants areidentified by differences in pattern among the DNAs of the sample set.

Exons of the candidate genes were amplified by PCR from the DNAs of 61related FAP patients and a control set of 12 normal individuals. Thefive exons from DP1 revealed no unique conformers in the FAP patients,although common conformers were observed with exons 2 and 3 in someindividuals of both affected and control sets, indicating the presenceof DNA sequence polymorphisms. Likewise, none of the five exons of SRP19revealed unique conformers in DNA from FAP patients in the test panel.

Testing of exons 1 through 14 and primer sets A through N of exon 15 ofthe DP2.5 gene, however, revealed variant conformers specific to FAPpatients in exons 7, 8, 10, 11, and 15. These variants were in theunrelated patients 3746, 3460, 3827, 3712, and 3751, respectively. ThePCR-SSCP procedure was repeated for each of these exons in the fiveaffected individuals and in an expanded set of 48 normal controls. Thevariant bands were reproducible in the FAP patients but were notobserved in any of the control DNA samples. Additional variantconformers in exons 11 and 15 of the DP2.5 gene were seen; however, eachof these was found in both the affected and control DNA sets. The fivesets of conformers unique to the FAP patients were sequenced todetermine the nucleotide changes responsible for their alteredmobillties. The normal conformers from the host individuals weresequenced also. Bands were cut from the dried acrylamide gels, and theDNA was eluted. PCR amplification of these DNAs provided template forsequencing.

The sequences of the unique conformers from exons 7, 8, 10, and 11 ofDP2.5 revealed dramatic mutations in the DP2.5 gene. The sequence of thenew mutation creating the exon 7 conformer in patient 3746 was shown tocontain a deletion of two adjacent nucleotides, at positions 730 and 731in the cDNA sequence .[.(FIG. 7).]. .Iadd.FIGS. 7A-7W (SEQ ID NO:1).Iaddend.. The normal sequence at this splice junction is CAGGGTCA(intronic sequence underlined), with the intron-exon boundary betweenthe two repetitions of AG. The mutant allele in this patient has thesequence CAGGTCA. Although this exchange is at the 5' splice site,comparison with known consensus sequences of splice junctions wouldsuggest that a functional splice junction is maintained. If this newsplice junction were functional, the mutation would introduce aframeshift that creates a stop codon 15 nueleotides downstream. If thenew splice junction were not functional, messenger processing would besignificantly altered.

To confirm the 2-base deletion, the PCR product from FAP patient 3746and a control DNA were electrophoresed on an acrylamide-urea denaturinggel, along with the products of a sequencing reaction. The sample frompatient 3746 showed two bands differing in size by 2 nucleotides, withthe larger band identical in mobility to the control sample; this resultwas independent confirmation that patient 3746 is heterozygous for a 2bp deletion.

The unique conformer found in exon 8 of patient 3460 was found to carrya C-T transition, at position 904 in the cDNA sequence of DP2.5 (shownin FIG. 7), which replaced the normal sequence of CGA with TGA. Thispoint mutation, when read in frame, results in a stop codon replacingthe normal arginine codon. This single-base change had occurred withinthe context of a CG dimer, a potential hot spot for mutation (Barker etal., 1984).

The conformer unique to FAP patient 3827 in exon 10 was found to containa deletion of one nucleotide (1367, 1368, or 1369) when compared to thenormal sequence found in the other bands on the SSCP gel. This deletion,occurring within a set of three T's, changed the sequence from CTTTCA toCTTCA; this 1 base frameshift creates a downstream stop within 30 bases.The PCR product amplified from this patient's DNA also waselectrophoresed on an aerylamide-urea denaturing along with the PCRproduct from a control DNA and products from a sequencing reaction. Thepatient's PCR product showed two bands differing by 1 bp in length, withthe larger identical in mobility to the PCR product from the normal DNA;this result confirmed the presence of a 1 bp deletion in patient 3827.

Sequence analysis of the variant conformer of exon 11 from patient 3712revealed the substitution of a T by a G at position 1500, changing thenormal tyrosine codon to a stop codon.

The pair of conformers observed in exon 15 of the DP2.5 gene for FAPpatient 3751 also was sequenced. These conformers were found to carry anucleotide substitution of C to G at position 5253, the third base of avaline codon. No amino acid change resulted from this substitution,suggesting that this conformer reflects a genetically silentpolymorphism.

The observation of distinct inactivating mutations in the DP2.5 gene infour unrelated patients strongly suggested that DP2.5 is the geneinvolved in FAP. These mutations are summarized in Table IIA.

EXAMPLE 10

This example demonstrates that the mutations identified in the DP2.5(APC) gene segregate with the FAP phenotype.

Patient 3746, described above as carrying an APC allele with aframeshift mutation, is an affected offspring of two normal parents.Colonoscopy revealed no polyps in either parent nor among the patient'sthree siblings.

DNA samples from both parents, from the patient's wife, and from theirthree children were examined. SSCP analysis of DNA from both of thepatient's parents displayed the normal pattern of conformers for exon 7,as did DNA from the patients's wife and one of his offspring. The twoother children, however, displayed the same new conformers as theiraffected father. Testing of the patient and his parents with highlypolymorphic VNTR (variable number of tandem repeat) markers showed a99.98% likelihood that they are his biological parents.

These observations confirmed that this novel conformer, known to reflecta 2 bp deletion mutation in the DP2.5 gene, appeared spontaneously withFAP in this pedigree and was transmitted to two of the children of theaffected individual.

EXAMPLE 11

This example demonstrates polymorphisms in the APC gene which appear tobe unrelated to disease (FAP).

Sequencing of variant conformers found among controls as well asindividuals with APC has revealed the following polymorphisms in the APCgene: first, in exon 11, at position 1458, a substitution of T to Ccreating an RsaI restriction site but no amino acid change; and second,in exon 15, at positions 5037 and 5271, substitutions of A to G and G toT, respectively, neither resulting in amino acid substitutions. Thesenucleotide polymorphisms in the APC gene sequence may be useful fordiagnostic purposes.

EXAMPLE 12

This example shows the structure of the APC gene.

The structure of the APC gene is schematically shown in FIG. 8, withflanking intron sequences indicated .Iadd.(SEQ ID NOS: 11-38).Iaddend..

The continuity of the very large (6.5 kb), most 3' exon in DP2.5 wasshown in two ways. First, inverse PCR with primers spanning the entirelength of this exon revealed no divergence of the cDNA sequence from thegenomic sequence. Second, PCR amplification with converging primersplaced at intervals along the exon generated products of the same sizewhether amplified from the originally isolated cDNA, cDNA from varioustissues, or genomie template. Two forms of exon 9 were found in DP2.5:one is the complete exon; and the other, labeled exon 9A, is the resultof a splice into the interior of the exon that deletes bases 934 to 1236in the mRNA and removes 101 amino acids from the predicted protein (see.[.FIG. 7.]. .Iadd.SEQ ID NOS: 1 & 2).Iaddend..

EXAMPLE 13

This example demonstrates the mapping of the FAP deletions with respectto the APC exons.

Somatie cell hybrids carrying the segregated chromosomes 5 from the 100kb (HHW1291) and 260 kb (HHW1155) deletion patients were used todetermine the distribution of the APC genes exons across the deletions.DNAs from these cell lines were used as template, along with genomie DNAfrom a normal control, for PCR-based amplification of the APC exons.

PCR analysis of the hybrids from the 260 kb deletion of patient 3214showed that all but one (exon 1) of the APC exons are removed by thisdeletion. PCR analysis of the somatie cell hybrid HHW1291, carrying thechromosome 5 homolog with the 100 kb deletion from patient 3824,revealed that exons 1 through 9 are present but exons 10 through 15 aremissing. This result placed the deletion breakpoint either between exons9 and 10 or within exon 10.

EXAMPLE 14

This example demonstrates the expression of alternately spliced APCmessenger in normal tissues and in cancer cell lines.

Tissues that express the APC gene were identified by PCR amplificationof cDNA made to mRNA with primers located within adjacent APC exons. Inaddition, PCR primers that flank the alternatively spliced exon 9 werechosen so that the expression pattern of both splice forms could beassessed. All tissue types tested (brain, lung, aorta, spleen, heart,kidney, liver, stomach, placenta, and eolonie mueosa) and cultured celllines (lymphoblasts, HL60, and ehorioeareinoma) expressed both spliceforms of the APC gene. We note, however, that expression by lymphocytesnormally residing in some tissues, including colon, prevents unequivocalassessment of expression. The large mRNA, containing the complete exon 9rather than only exon 9A, appears to be the more abundant message.

Northern analysis of poly(A)-selected RNA from lymphoblasts revealed asingle band of approximately 10 kb, consistent with the size of thesequenced cDNA.

EXAMPLE 15

This example discusses structural features of the APC protein predictedfrom the sequence.

The cDNA consensus sequence of APC predicts that the longer, moreabundant form of the message codes for a .[.2842 or 28444.]. .Iadd.2843.Iaddend.amino acid peptide with a mass of 311.8 kd. This predicted APCpeptide was compared with the current data bases of protein and DNAsequences using both Intelligenetics and GCG software packages. No geneswith a high degree of amino acid sequence similarity were found.Although many short (approximately 20 amino acid) regions of sequencesimilarity were uncovered, none was sufficiently strong to reveal which,if any, might represent functional hornology. Interestingly, multiplesimilarities to myosins and keratins did appear. The APC gene also wasscanned for sequence motifs of known function; although multipleglycosylation, phosphorylation, and myristoylation sites were seen,their significance is uncertain.

Analysis of the APC peptide sequence did identify features important inconsidering potential protein structure. Hydropathy plots (Kyte andDoolittle, J. Mol. Biol. Vol. 157, pp. 105-132 (1982)) indicate that theAPC protein is notably hydrophilic. No hydrophobic domains suggesting asignal peptide or a membrane-spanning domain were found. Analysis of thefirst 1000 residues indicates that α-helical rods may form (Cohen andParry, Trends Biochem, Sci. Vol. 77, pp. 245-248 (1986); there is ascarcity of proline residues and, there are a number of regionscontaining heptad repeats (apolar-X-X-apolar-X-X-X). Interestingly, inexon 9A, the deleted form of exon 9, two heptad repeat regions arereconnected in the proper heptad repeat frame, deleting the interveningpeptide region. After the first 1000 residues, the high proline contentof the remainder of the peptide suggests a compact rather than arod-like structure.

The most prominent feature of the second 1000 residues is a 20 aminoacid repeat that is iterated seven times with semiregular spacing (Table4). The intervening sequences between the seven repeat regions contained114, 116, 151, 205, 107, and 58 amino acids, respectively. Finally,residues 2200-24000 contain a 200 amino acid basic domain.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 102                                           - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 8532 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: DP2.5(APC)                                               -   (viii) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATGGCTGCAG CTTCATATGA TCAGTTGTTA AAGCAAGTTG AGGCACTGAA GA - #TGGAGAAC         60                                                                          - TCAAATCTTC GACAAGAGCT AGAAGATAAT TCCAATCATC TTACAAAACT GG - #AAACTGAG        120                                                                          - GCATCTAATA TGAAGGAAGT ACTTAAACAA CTACAAGGAA GTATTGAAGA TG - #AAGCTATG        180                                                                          - GCTTCTTCTG GACAGATTGA TTTATTAGAG CGTCTTAAAG AGCTTAACTT AG - #ATAGCAGT        240                                                                          - AATTTCCCTG GAGTAAAACT GCGGTCAAAA ATGTCCCTCC GTTCTTATGG AA - #GCCGGGAA        300                                                                          - GGATCTGTAT CAAGCCGTTC TGGAGAGTGC AGTCCTGTTC CTATGGGTTC AT - #TTCCAAGA        360                                                                          - AGAGGGTTTG TAAATGGAAG CAGAGAAAGT ACTGGATATT TAGAAGAACT TG - #AGAAAGAG        420                                                                          - AGGTCATTGC TTCTTGCTGA TCTTGACAAA GAAGAAAAGG AAAAAGACTG GT - #ATTACGCT        480                                                                          - CAACTTCAGA ATCTCACTAA AAGAATAGAT AGTCTTCCTT TAACTGAAAA TT - #TTTCCTTA        540                                                                          - CAAACAGATA TGACCAGAAG GCAATTGGAA TATGAAGCAA GGCAAATCAG AG - #TTGCGATG        600                                                                          - GAAGAACAAC TAGGTACCTG CCAGGATATG GAAAAACGAG CACAGCGAAG AA - #TAGCCAGA        660                                                                          - ATTCAGCAAA TCGAAAAGGA CATACTTCGT ATACGACAGC TTTTACAGTC CC - #AAGCAACA        720                                                                          - GAAGCAGAGA GGTCATCTCA GAACAAGCAT GAAACCGGCT CACATGATGC TG - #AGCGGCAG        780                                                                          - AATGAAGGTC AAGGAGTGGG AGAAATCAAC ATGGCAACTT CTGGTAATGG TC - #AGGGTTCA        840                                                                          - ACTACACGAA TGGACCATGA AACAGCCAGT GTTTTGAGTT CTAGTAGCAC AC - #ACTCTGCA        900                                                                          - CCTCGAAGGC TGACAAGTCA TCTGGGAACC AAGGTGGAAA TGGTGTATTC AT - #TGTTGTCA        960                                                                          - ATGCTTGGTA CTCATGATAA GGATGATATG TCGCGAACTT TGCTAGCTAT GT - #CTAGCTCC       1020                                                                          - CAAGACAGCT GTATATCCAT GCGACAGTCT GGATGTCTTC CTCTCCTCAT CC - #AGCTTTTA       1080                                                                          - CATGGCAATG ACAAAGACTC TGTATTGTTG GGAAATTCCC GGGGCAGTAA AG - #AGGCTCGG       1140                                                                          - GCCAGGGCCA GTGCAGCACT CCACAACATC ATTCACTCAC AGCCTGATGA CA - #AGAGAGGC       1200                                                                          - AGGCGTGAAA TCCGAGTCCT TCATCTTTTG GAACAGATAC GCGCTTACTG TG - #AAACCTGT       1260                                                                          - TGGGAGTGGC AGGAAGCTCA TGAACCAGGC ATGGACCAGG ACAAAAATCC AA - #TGCCAGCT       1320                                                                          - CCTGTTGAAC ATCAGATCTG TCCTGCTGTG TGTGTTCTAA TGAAACTTTC AT - #TTGATGAA       1380                                                                          - GAGCATAGAC ATGCAATGAA TGAACTAGGG GGACTACAGG CCATTGCAGA AT - #TATTGCAA       1440                                                                          - GTGGACTGTG AAATGTACGG GCTTACTAAT GACCACTACA GTATTACACT AA - #GACGATAT       1500                                                                          - GCTGGAATGG CTTTGACAAA CTTGACTTTT GGAGATGTAG CCAACAAGGC TA - #CGCTATGC       1560                                                                          - TCTATGAAAG GCTGCATGAG AGCACTTGTG GCCCAACTAA AATCTGAAAG TG - #AAGACTTA       1620                                                                          - CAGCAGGTTA TTGCAAGTGT TTTGAGGAAT TTGTCTTGGC GAGCAGATGT AA - #ATAGTAAA       1680                                                                          - AAGACGTTGC GAGAAGTTGG AAGTGTGAAA GCATTGATGG AATGTGCTTT AG - #AAGTTAAA       1740                                                                          - AAGGAATCAA CCCTCAAAAG CGTATTGAGT GCCTTATGGA ATTTGTCAGC AC - #ATTGCACT       1800                                                                          - GAGAATAAAG CTGATATATG TGCTGTAGAT GGTGCACTTG CATTTTTGGT TG - #GCACTCTT       1860                                                                          - ACTTACCGGA GCCAGACAAA CACTTTAGCC ATTATTGAAA GTGGAGGTGG GA - #TATTACGG       1920                                                                          - AATGTGTCCA GCTTGATAGC TACAAATGAG GACCACAGGC AAATCCTAAG AG - #AGAACAAC       1980                                                                          - TGTCTACAAA CTTTATTACA ACACTTAAAA TCTCATAGTT TGACAATAGT CA - #GTAATGCA       2040                                                                          - TGTGGAACTT TGTGGAATCT CTCAGCAAGA AATCCTAAAG ACCAGGAAGC AT - #TATGGGAC       2100                                                                          - ATGGGGGCAG TTAGCATGCT CAAGAACCTC ATTCATTCAA AGCACAAAAT GA - #TTGCTATG       2160                                                                          - GGAAGTGCTG CAGCTTTAAG GAATCTCATG GCAAATAGGC CTGCGAAGTA CA - #AGGATGCC       2220                                                                          - AATATTATGT CTCCTGGCTC AAGCTTGCCA TCTCTTCATG TTAGGAAACA AA - #AAGCCCTA       2280                                                                          - GAAGCAGAAT TAGATGCTCA GCACTTATCA GAAACTTTTG ACAATATAGA CA - #ATTTAAGT       2340                                                                          - CCCAAGGCAT CTCATCGTAG TAAGCAGAGA CACAAGCAAA GTCTCTATGG TG - #ATTATGTT       2400                                                                          - TTTGACACCA ATCGACATGA TGATAATAGG TCAGACAATT TTAATACTGG CA - #ACATGACT       2460                                                                          - GTCCTTTCAC CATATTTGAA TACTACAGTG TTACCCAGCT CCTCTTCATC AA - #GAGGAAGC       2520                                                                          - TTAGATAGTT CTCGTTCTGA AAAAGATAGA AGTTTGGAGA GAGAACGCGG AA - #TTGGTCTA       2580                                                                          - GGCAACTACC ATCCAGCAAC AGAAAATCCA GGAACTTCTT CAAAGCGAGG TT - #TGCAGATC       2640                                                                          - TCCACCACTG CAGCCCAGAT TGCCAAAGTC ATGGAAGAAG TGTCAGCCAT TC - #ATACCTCT       2700                                                                          - CAGGAAGACA GAAGTTCTGG GTCTACCACT GAATTACATT GTGTGACAGA TG - #AGAGAAAT       2760                                                                          - GCACTTAGAA GAAGCTCTGC TGCCCATACA CATTCAAACA CTTACAATTT CA - #CTAAGTCG       2820                                                                          - GAAAATTCAA ATAGGACATG TTCTATGCCT TATGCCAAAT TAGAATACAA GA - #GATCTTCA       2880                                                                          - AATGATAGTT TAAATAGTGT CAGTAGTAGT GATGGTTATG GTAAAAGAGG TC - #AAATGAAA       2940                                                                          - CCCTCGATTG AATCCTATTC TGAAGATGAT GAAAGTAAGT TTTGCAGTTA TG - #GTCAATAC       3000                                                                          - CCAGCCGACC TAGCCCATAA AATACATAGT GCAAATCATA TGGATGATAA TG - #ATGGAGAA       3060                                                                          - CTAGATACAC CAATAAATTA TAGTCTTAAA TATTCAGATG AGCAGTTGAA CT - #CTGGAAGG       3120                                                                          - CAAAGTCCTT CACAGAATGA AAGATGGGCA AGACCCAAAC ACATAATAGA AG - #ATGAAATA       3180                                                                          - AAACAAAGTG AGCAAAGACA ATCAAGGAAT CAAAGTACAA CTTATCCTGT TT - #ATACTGAG       3240                                                                          - AGCACTGATG ATAAACACCT CAAGTTCCAA CCACATTTTG GACAGCAGGA AT - #GTGTTTCT       3300                                                                          - CCATACAGGT CACGGGGAGC CAATGGTTCA GAAACAAATC GAGTGGGTTC TA - #ATCATGGA       3360                                                                          - ATTAATCAAA ATGTAAGCCA GTCTTTGTGT CAAGAAGATG ACTATGAAGA TG - #ATAAGCCT       3420                                                                          - ACCAATTATA GTGAACGTTA CTCTGAAGAA GAACAGCATG AAGAAGAAGA GA - #GACCAACA       3480                                                                          - AATTATAGCA TAAAATATAA TGAAGAGAAA CGTCATGTGG ATCAGCCTAT TG - #ATTATAGT       3540                                                                          - TTAAAATATG CCACAGATAT TCCTTCATCA CAGAAACAGT CATTTTCATT CT - #CAAAGAGT       3600                                                                          - TCATCTGGAC AAAGCAGTAA AACCGAACAT ATGTCTTCAA GCAGTGAGAA TA - #CGTCCACA       3660                                                                          - CCTTCATCTA ATGCCAAGAG GCAGAATCAG CTCCATCCAA GTTCTGCACA GA - #GTAGAAGT       3720                                                                          - GGTCAGCCTC AAAAGGCTGC CACTTGCAAA GTTTCTTCTA TTAACCAAGA AA - #CAATACAG       3780                                                                          - ACTTATTGTG TAGAAGATAC TCCAATATGT TTTTCAAGAT GTAGTTCATT AT - #CATCTTTG       3840                                                                          - TCATCAGCTG AAGATGAAAT AGGATGTAAT CAGACGACAC AGGAAGCAGA TT - #CTGCTAAT       3900                                                                          - ACCCTGCAAA TAGCAGAAAT AAAAGAAAAG ATTGGAACTA GGTCAGCTGA AG - #ATCCTGTG       3960                                                                          - AGCGAAGTTC CAGCAGTGTC ACAGCACCCT AGAACCAAAT CCAGCAGACT GC - #AGGGTTCT       4020                                                                          - AGTTTATCTT CAGAATCAGC CAGGCACAAA GCTGTTGAAT TTTCTTCAGG AG - #CGAAATCT       4080                                                                          - CCCTCCAAAA GTGGTGCTCA GACACCCAAA AGTCCACCTG AACACTATGT TC - #AGGAGACC       4140                                                                          - CCACTCATGT TTAGCAGATG TACTTCTGTC AGTTCACTTG ATAGTTTTGA GA - #GTCGTTCG       4200                                                                          - ATTGCCAGCT CCGTTCAGAG TGAACCATGC AGTGGAATGG TAAGTGGCAT TA - #TAAGCCCC       4260                                                                          - AGTGATCTTC CAGATAGCCC TGGACAAACC ATGCCACCAA GCAGAAGTAA AA - #CACCTCCA       4320                                                                          - CCACCTCCTC AAACAGCTCA AACCAAGCGA GAAGTACCTA AAAATAAAGC AC - #CTACTGCT       4380                                                                          - GAAAAGAGAG AGAGTGGACC TAAGCAAGCT GCAGTAAATG CTGCAGTTCA GA - #GGGTCCAG       4440                                                                          - GTTCTTCCAG ATGCTGATAC TTTATTACAT TTTGCCACGG AAAGTACTCC AG - #ATGGATTT       4500                                                                          - TCTTGTTCAT CCAGCCTGAG TGCTCTGAGC CTCGATGAGC CATTTATACA GA - #AAGATGTG       4560                                                                          - GAATTAAGAA TAATGCCTCC AGTTCAGGAA AATGACAATG GGAATGAAAC AG - #AATCAGAG       4620                                                                          - CAGCCTAAAG AATCAAATGA AAACCAAGAG AAAGAGGCAG AAAAAACTAT TG - #ATTCTGAA       4680                                                                          - AAGGACCTAT TAGATGATTC AGATGATGAT GATATTGAAA TACTAGAAGA AT - #GTATTATT       4740                                                                          - TCTGCCATGC CAACAAAGTC ATCACGTAAA GCAAAAAAGC CAGCCCAGAC TG - #CTTCAAAA       4800                                                                          - TTACCTCCAC CTGTGGCAAG GAAACCAAGT CAGCTGCCTG TGTACAAACT TC - #TACCATCA       4860                                                                          - CAAAACAGGT TGCAACCCCA AAAGCATGTT AGTTTTACAC CGGGGGATGA TA - #TGCCACGG       4920                                                                          - GTGTATTGTG TTGAAGGGAC ACCTATAAAC TTTTCCACAG CTACATCTCT AA - #GTGATCTA       4980                                                                          - ACAATCGAAT CCCCTCCAAA TGAGTTAGCT GCTGGAGAAG GAGTTAGAGG AG - #GAGCACAG       5040                                                                          - TCAGGTGAAT TTGAAAAACG AGATACCATT CCTACAGAAG GCAGAAGTAC AG - #ATGAGGCT       5100                                                                          - CAAGGAGGAA AAACCTCATC TGTAACCATA CCTGAATTGG ATGACAATAA AG - #CAGAGGAA       5160                                                                          - GGTGATATTC TTGCAGAATG CATTAATTCT GCTATGCCCA AAGGGAAAAG TC - #ACAAGCCT       5220                                                                          - TTCCGTGTGA AAAAGATAAT GGACCAGGTC CAGCAAGCAT CTGCGTCGTC TT - #CTGCACCC       5280                                                                          - AACAAAAATC AGTTAGATGG TAAGAAAAAG AAACCAACTT CACCAGTAAA AC - #CTATACCA       5340                                                                          - CAAAATACTG AATATAGGAC ACGTGTAAGA AAAAATGCAG ACTCAAAAAA TA - #ATTTAAAT       5400                                                                          - GCTGAGAGAG TTTTCTCAGA CAACAAAGAT TCAAAGAAAC AGAATTTGAA AA - #ATAATTCC       5460                                                                          - AAGGACTTCA ATGATAAGCT CCCAAATAAT GAAGATAGAG TCAGAGGAAG TT - #TTGCTTTT       5520                                                                          - GATTCACCTC ATCATTACAC GCCTATTGAA GGAACTCCTT ACTGTTTTTC AC - #GAAATGAT       5580                                                                          - TCTTTGAGTT CTCTAGATTT TGATGATGAT GATGTTGACC TTTCCAGGGA AA - #AGGCTGAA       5640                                                                          - TTAAGAAAGG CAAAAGAAAA TAAGGAATCA GAGGCTAAAG TTACCAGCCA CA - #CAGAACTA       5700                                                                          - ACCTCCAACC AACAATCAGC TAATAAGACA CAAGCTATTG CAAAGCAGCC AA - #TAAATCGA       5760                                                                          - GGTCAGCCTA AACCCATACT TCAGAAACAA TCCACTTTTC CCCAGTCATC CA - #AAGACATA       5820                                                                          - CCAGACAGAG GGGCAGCAAC TGATGAAAAG TTACAGAATT TTGCTATTGA AA - #ATACTCCA       5880                                                                          - GTTTGCTTTT CTCATAATTC CTCTCTGAGT TCTCTCAGTG ACATTGACCA AG - #AAAACAAC       5940                                                                          - AATAAAGAAA ATGAACCTAT CAAAGAGACT GAGCCCCCTG ACTCACAGGG AG - #AACCAAGT       6000                                                                          - AAACCTCAAG CATCAGGCTA TGCTCCTAAA TCATTTCATG TTGAAGATAC CC - #CAGTTTGT       6060                                                                          - TTCTCAAGAA ACAGTTCTCT CAGTTCTCTT AGTATTGACT CTGAAGATGA CC - #TGTTGCAG       6120                                                                          - GAATGTATAA GCTCCGCAAT GCCAAAAAAG AAAAAGCCTT CAAGACTCAA GG - #GTGATAAT       6180                                                                          - GAAAAACATA GTCCCAGAAA TATGGGTGGC ATATTAGGTG AAGATCTGAC AC - #TTGATTTG       6240                                                                          - AAAGATATAC AGAGACCAGA TTCAGAACAT GGTCTATCCC CTGATTCAGA AA - #ATTTTGAT       6300                                                                          - TGGAAAGCTA TTCAGGAAGG TGCAAATTCC ATAGTAAGTA GTTTACATCA AG - #CTGCTGCT       6360                                                                          - GCTGCATGTT TATCTAGACA AGCTTCGTCT GATTCAGATT CCATCCTTTC CC - #TGAAATCA       6420                                                                          - GGAATCTCTC TGGGATCACC ATTTCATCTT ACACCTGATC AAGAAGAAAA AC - #CCTTTACA       6480                                                                          - AGTAATAAAG GCCCACGAAT TCTAAAACCA GGGGAGAAAA GTACATTGGA AA - #CTAAAAAG       6540                                                                          - ATAGAATCTG AAAGTAAAGG AATCAAAGGA GGAAAAAAAG TTTATAAAAG TT - #TGATTACT       6600                                                                          - GGAAAAGTTC GATCTAATTC AGAAATTTCA GGCCAAATGA AACAGCCCCT TC - #AAGCAAAC       6660                                                                          - ATGCCTTCAA TCTCTCGAGG CAGGACAATG ATTCATATTC CAGGAGTTCG AA - #ATAGCTCC       6720                                                                          - TCAAGTACAA GTCCTGTTTC TAAAAAAGGC CCACCCCTTA AGACTCCAGC CT - #CCAAAAGC       6780                                                                          - CCTAGTGAAG GTCAAACAGC CACCACTTCT CCTAGAGGAG CCAAGCCATC TG - #TGAAATCA       6840                                                                          - GAATTAAGCC CTGTTGCCAG GCAGACATCC CAAATAGGTG GGTCAAGTAA AG - #CACCTTCT       6900                                                                          - AGATCAGGAT CTAGAGATTC GACCCCTTCA AGACCTGCCC AGCAACCATT AA - #GTAGACCT       6960                                                                          - ATACAGTCTC CTGGCCGAAA CTCAATTTCC CCTGGTAGAA ATGGAATAAG TC - #CTCCTAAC       7020                                                                          - AAATTATCTC AACTTCCAAG GACATCATCC CCTAGTACTG CTTCAACTAA GT - #CCTCAGGT       7080                                                                          - TCTGGAAAAA TGTCATATAC ATCTCCAGGT AGACAGATGA GCCAACAGAA CC - #TTACCAAA       7140                                                                          - CAAACAGGTT TATCCAAGAA TGCCAGTAGT ATTCCAAGAA GTGAGTCTGC CT - #CCAAAGGA       7200                                                                          - CTAAATCAGA TGAATAATGG TAATGGAGCC AATAAAAAGG TAGAACTTTC TA - #GAATGTCT       7260                                                                          - TCAACTAAAT CAAGTGGAAG TGAATCTGAT AGATCAGAAA GACCTGTATT AG - #TACGCCAG       7320                                                                          - TCAACTTTCA TCAAAGAAGC TCCAAGCCCA ACCTTAAGAA GAAAATTGGA GG - #AATCTGCT       7380                                                                          - TCATTTGAAT CTCTTTCTCC ATCATCTAGA CCAGCTTCTC CCACTAGGTC CC - #AGGCACAA       7440                                                                          - ACTCCAGTTT TAAGTCCTTC CCTTCCTGAT ATGTCTCTAT CCACACATTC GT - #CTGTTCAG       7500                                                                          - GCTGGTGGAT GGCGAAAACT CCCACCTAAT CTCAGTCCCA CTATAGAGTA TA - #ATGATGGA       7560                                                                          - AGACCAGCAA AGCGCCATGA TATTGCACGG TCTCATTCTG AAAGTCCTTC TA - #GACTTCCA       7620                                                                          - ATCAATAGGT CAGGAACCTG GAAACGTGAG CACAGCAAAC ATTCATCATC CC - #TTCCTCGA       7680                                                                          - GTAAGCACTT GGAGAAGAAC TGGAAGTTCA TCTTCAATTC TTTCTGCTTC AT - #CAGAATCC       7740                                                                          - AGTGAAAAAG CAAAAAGTGA GGATGAAAAA CATGTGAACT CTATTTCAGG AA - #CCAAACAA       7800                                                                          - AGTAAAGAAA ACCAAGTATC CGCAAAAGGA ACATGGAGAA AAATAAAAGA AA - #ATGAATTT       7860                                                                          - TCTCCCACAA ATAGTACTTC TCAGACCGTT TCCTCAGGTG CTACAAATGG TG - #CTGAATCA       7920                                                                          - AAGACTCTAA TTTATCAAAT GGCACCTGCT GTTTCTAAAA CAGAGGATGT TT - #GGGTGAGA       7980                                                                          - ATTGAGGACT GTCCCATTAA CAATCCTAGA TCTGGAAGAT CTCCCACAGG TA - #ATACTCCC       8040                                                                          - CCGGTGATTG ACAGTGTTTC AGAAAAGGCA AATCCAAACA TTAAAGATTC AA - #AAGATAAT       8100                                                                          - CAGGCAAAAC AAAATGTGGG TAATGGCAGT GTTCCCATGC GTACCGTGGG TT - #TGGAAAAT       8160                                                                          - CGCCTGAACT CCTTTATTCA GGTGGATGCC CCTGACCAAA AAGGAACTGA GA - #TAAAACCA       8220                                                                          - GGACAAAATA ATCCTGTCCC TGTATCAGAG ACTAATGAAA GTTCTATAGT GG - #AACGTACC       8280                                                                          - CCATTCAGTT CTAGCAGCTC AAGCAAACAC AGTTCACCTA GTGGGACTGT TG - #CTGCCAGA       8340                                                                          - GTGACTCCTT TTAATTACAA CCCAAGCCCT AGGAAAAGCA GCGCAGATAG CA - #CTTCAGCT       8400                                                                          - CGGCCATCTC AGATCCCAAC TCCAGTGAAT AACAACACAA AGAAGCGAGA TT - #CCAAAACT       8460                                                                          - GACAGCACAG AATCCAGTGG AACCCAAAGT CCTAAGCGCC ATTCTGGGTC TT - #ACCTTGTG       8520                                                                          #     8532                                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 2843 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Ala Ala Ala Ser Tyr Asp Gln Leu Leu Ly - #s Gln Val Glu Ala Leu         #                15                                                           - Lys Met Glu Asn Ser Asn Leu Arg Gln Glu Le - #u Glu Asp Asn Ser Asn         #            30                                                               - His Leu Thr Lys Leu Glu Thr Glu Ala Ser As - #n Met Lys Glu Val Leu         #        45                                                                   - Lys Gln Leu Gln Gly Ser Ile Glu Asp Glu Al - #a Met Ala Ser Ser Gly         #    60                                                                       - Gln Ile Asp Leu Leu Glu Arg Leu Lys Glu Le - #u Asn Leu Asp Ser Ser         #80                                                                           - Asn Phe Pro Gly Val Lys Leu Arg Ser Lys Me - #t Ser Leu Arg Ser Tyr         #                95                                                           - Gly Ser Arg Glu Gly Ser Val Ser Ser Arg Se - #r Gly Glu Cys Ser Pro         #           110                                                               - Val Pro Met Gly Ser Phe Pro Arg Arg Gly Ph - #e Val Asn Gly Ser Arg         #       125                                                                   - Glu Ser Thr Gly Tyr Leu Glu Glu Leu Glu Ly - #s Glu Arg Ser Leu Leu         #   140                                                                       - Leu Ala Asp Leu Asp Lys Glu Glu Lys Glu Ly - #s Asp Trp Tyr Tyr Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Leu Gln Asn Leu Thr Lys Arg Ile Asp Se - #r Leu Pro Leu Thr Glu         #               175                                                           - Asn Phe Ser Leu Gln Thr Asp Met Thr Arg Ar - #g Gln Leu Glu Tyr Glu         #           190                                                               - Ala Arg Gln Ile Arg Val Ala Met Glu Glu Gl - #n Leu Gly Thr Cys Gln         #       205                                                                   - Asp Met Glu Lys Arg Ala Gln Arg Arg Ile Al - #a Arg Ile Gln Gln Ile         #   220                                                                       - Glu Lys Asp Ile Leu Arg Ile Arg Gln Leu Le - #u Gln Ser Gln Ala Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Glu Ala Glu Arg Ser Ser Gln Asn Lys His Gl - #u Thr Gly Ser His Asp         #               255                                                           - Ala Glu Arg Gln Asn Glu Gly Gln Gly Val Gl - #y Glu Ile Asn Met Ala         #           270                                                               - Thr Ser Gly Asn Gly Gln Gly Ser Thr Thr Ar - #g Met Asp His Glu Thr         #       285                                                                   - Ala Ser Val Leu Ser Ser Ser Ser Thr His Se - #r Ala Pro Arg Arg Leu         #   300                                                                       - Thr Ser His Leu Gly Thr Lys Val Glu Met Va - #l Tyr Ser Leu Leu Ser         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Met Leu Gly Thr His Asp Lys Asp Asp Met Se - #r Arg Thr Leu Leu Ala         #               335                                                           - Met Ser Ser Ser Gln Asp Ser Cys Ile Ser Me - #t Arg Gln Ser Gly Cys         #           350                                                               - Leu Pro Leu Leu Ile Gln Leu Leu His Gly As - #n Asp Lys Asp Ser Val         #       365                                                                   - Leu Leu Gly Asn Ser Arg Gly Ser Lys Glu Al - #a Arg Ala Arg Ala Ser         #   380                                                                       - Ala Ala Leu His Asn Ile Ile His Ser Gln Pr - #o Asp Asp Lys Arg Gly         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Arg Arg Glu Ile Arg Val Leu His Leu Leu Gl - #u Gln Ile Arg Ala Tyr         #               415                                                           - Cys Glu Thr Cys Trp Glu Trp Gln Glu Ala Hi - #s Glu Pro Gly Met Asp         #           430                                                               - Gln Asp Lys Asn Pro Met Pro Ala Pro Val Gl - #u His Gln Ile Cys Pro         #       445                                                                   - Ala Val Cys Val Leu Met Lys Leu Ser Phe As - #p Glu Glu His Arg His         #   460                                                                       - Ala Met Asn Glu Leu Gly Gly Leu Gln Ala Il - #e Ala Glu Leu Leu Gln         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Val Asp Cys Glu Met Tyr Gly Leu Thr Asn As - #p His Tyr Ser Ile Thr         #               495                                                           - Leu Arg Arg Tyr Ala Gly Met Ala Leu Thr As - #n Leu Thr Phe Gly Asp         #           510                                                               - Val Ala Asn Lys Ala Thr Leu Cys Ser Met Ly - #s Gly Cys Met Arg Ala         #       525                                                                   - Leu Val Ala Gln Leu Lys Ser Glu Ser Glu As - #p Leu Gln Gln Val Ile         #   540                                                                       - Ala Ser Val Leu Arg Asn Leu Ser Trp Arg Al - #a Asp Val Asn Ser Lys         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Lys Thr Leu Arg Glu Val Gly Ser Val Lys Al - #a Leu Met Glu Cys Ala         #               575                                                           - Leu Glu Val Lys Lys Glu Ser Thr Leu Lys Se - #r Val Leu Ser Ala Leu         #           590                                                               - Trp Asn Leu Ser Ala His Cys Thr Glu Asn Ly - #s Ala Asp Ile Cys Ala         #       605                                                                   - Val Asp Gly Ala Leu Ala Phe Leu Val Gly Th - #r Leu Thr Tyr Arg Ser         #   620                                                                       - Gln Thr Asn Thr Leu Ala Ile Ile Glu Ser Gl - #y Gly Gly Ile Leu Arg         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Asn Val Ser Ser Leu Ile Ala Thr Asn Glu As - #p His Arg Gln Ile Leu         #               655                                                           - Arg Glu Asn Asn Cys Leu Gln Thr Leu Leu Gl - #n His Leu Lys Ser His         #           670                                                               - Ser Leu Thr Ile Val Ser Asn Ala Cys Gly Th - #r Leu Trp Asn Leu Ser         #       685                                                                   - Ala Arg Asn Pro Lys Asp Gln Glu Ala Leu Tr - #p Asp Met Gly Ala Val         #   700                                                                       - Ser Met Leu Lys Asn Leu Ile His Ser Lys Hi - #s Lys Met Ile Ala Met         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Gly Ser Ala Ala Ala Leu Arg Asn Leu Met Al - #a Asn Arg Pro Ala Lys         #               735                                                           - Tyr Lys Asp Ala Asn Ile Met Ser Pro Gly Se - #r Ser Leu Pro Ser Leu         #           750                                                               - His Val Arg Lys Gln Lys Ala Leu Glu Ala Gl - #u Leu Asp Ala Gln His         #       765                                                                   - Leu Ser Glu Thr Phe Asp Asn Ile Asp Asn Le - #u Ser Pro Lys Ala Ser         #   780                                                                       - His Arg Ser Lys Gln Arg His Lys Gln Ser Le - #u Tyr Gly Asp Tyr Val         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Phe Asp Thr Asn Arg His Asp Asp Asn Arg Se - #r Asp Asn Phe Asn Thr         #               815                                                           - Gly Asn Met Thr Val Leu Ser Pro Tyr Leu As - #n Thr Thr Val Leu Pro         #           830                                                               - Ser Ser Ser Ser Ser Arg Gly Ser Leu Asp Se - #r Ser Arg Ser Glu Lys         #       845                                                                   - Asp Arg Ser Leu Glu Arg Glu Arg Gly Ile Gl - #y Leu Gly Asn Tyr His         #   860                                                                       - Pro Ala Thr Glu Asn Pro Gly Thr Ser Ser Ly - #s Arg Gly Leu Gln Ile         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Ser Thr Thr Ala Ala Gln Ile Ala Lys Val Me - #t Glu Glu Val Ser Ala         #               895                                                           - Ile His Thr Ser Gln Glu Asp Arg Ser Ser Gl - #y Ser Thr Thr Glu Leu         #           910                                                               - His Cys Val Thr Asp Glu Arg Asn Ala Leu Ar - #g Arg Ser Ser Ala Ala         #       925                                                                   - His Thr His Ser Asn Thr Tyr Asn Phe Thr Ly - #s Ser Glu Asn Ser Asn         #   940                                                                       - Arg Thr Cys Ser Met Pro Tyr Ala Lys Leu Gl - #u Tyr Lys Arg Ser Ser         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Asn Asp Ser Leu Asn Ser Val Ser Ser Ser As - #p Gly Tyr Gly Lys Arg         #               975                                                           - Gly Gln Met Lys Pro Ser Ile Glu Ser Tyr Se - #r Glu Asp Asp Glu Ser         #           990                                                               - Lys Phe Cys Ser Tyr Gly Gln Tyr Pro Ala As - #p Leu Ala His Lys Ile         #     10050                                                                   - His Ser Ala Asn His Met Asp Asp Asn Asp Gl - #y Glu Leu Asp Thr Pro         # 10205                                                                       - Ile Asn Tyr Ser Leu Lys Tyr Ser Asp Glu Gl - #n Leu Asn Ser Gly Arg         #              10405  0                                                       - Gln Ser Pro Ser Gln Asn Glu Arg Trp Ala Ar - #g Pro Lys His Ile Ile         #             10550                                                           - Glu Asp Glu Ile Lys Gln Ser Glu Gln Arg Gl - #n Ser Arg Asn Gln Ser         #         10705                                                               - Thr Thr Tyr Pro Val Tyr Thr Glu Ser Thr As - #p Asp Lys His Leu Lys         #     10850                                                                   - Phe Gln Pro His Phe Gly Gln Gln Glu Cys Va - #l Ser Pro Tyr Arg Ser         # 11005                                                                       - Arg Gly Ala Asn Gly Ser Glu Thr Asn Arg Va - #l Gly Ser Asn His Gly         #              11205  0                                                       - Ile Asn Gln Asn Val Ser Gln Ser Leu Cys Gl - #n Glu Asp Asp Tyr Glu         #             11350                                                           - Asp Asp Lys Pro Thr Asn Tyr Ser Glu Arg Ty - #r Ser Glu Glu Glu Gln         #         11505                                                               - His Glu Glu Glu Glu Arg Pro Thr Asn Tyr Se - #r Ile Lys Tyr Asn Glu         #     11650                                                                   - Glu Lys Arg His Val Asp Gln Pro Ile Asp Ty - #r Ser Leu Lys Tyr Ala         # 11805                                                                       - Thr Asp Ile Pro Ser Ser Gln Lys Gln Ser Ph - #e Ser Phe Ser Lys Ser         #              12005  0                                                       - Ser Ser Gly Gln Ser Ser Lys Thr Glu His Me - #t Ser Ser Ser Ser Glu         #             12150                                                           - Asn Thr Ser Thr Pro Ser Ser Asn Ala Lys Ar - #g Gln Asn Gln Leu His         #         12305                                                               - Pro Ser Ser Ala Gln Ser Arg Ser Gly Gln Pr - #o Gln Lys Ala Ala Thr         #     12450                                                                   - Cys Lys Val Ser Ser Ile Asn Gln Glu Thr Il - #e Gln Thr Tyr Cys Val         # 12605                                                                       - Glu Asp Thr Pro Ile Cys Phe Ser Arg Cys Se - #r Ser Leu Ser Ser Leu         #              12805  0                                                       - Ser Ser Ala Glu Asp Glu Ile Gly Cys Asn Gl - #n Thr Thr Gln Glu Ala         #             12950                                                           - Asp Ser Ala Asn Thr Leu Gln Ile Ala Glu Il - #e Lys Glu Lys Ile Gly         #         13105                                                               - Thr Arg Ser Ala Glu Asp Pro Val Ser Glu Va - #l Pro Ala Val Ser Gln         #     13250                                                                   - His Pro Arg Thr Lys Ser Ser Arg Leu Gln Gl - #y Ser Ser Leu Ser Ser         # 13405                                                                       - Glu Ser Ala Arg His Lys Ala Val Glu Phe Se - #r Ser Gly Ala Lys Ser         #              13605  0                                                       - Pro Ser Lys Ser Gly Ala Gln Thr Pro Lys Se - #r Pro Pro Glu His Tyr         #             13750                                                           - Val Gln Glu Thr Pro Leu Met Phe Ser Arg Cy - #s Thr Ser Val Ser Ser         #         13905                                                               - Leu Asp Ser Phe Glu Ser Arg Ser Ile Ala Se - #r Ser Val Gln Ser Glu         #     14050                                                                   - Pro Cys Ser Gly Met Val Ser Gly Ile Ile Se - #r Pro Ser Asp Leu Pro         # 14205                                                                       - Asp Ser Pro Gly Gln Thr Met Pro Pro Ser Ar - #g Ser Lys Thr Pro Pro         #              14405  0                                                       - Pro Pro Pro Gln Thr Ala Gln Thr Lys Arg Gl - #u Val Pro Lys Asn Lys         #             14550                                                           - Ala Pro Thr Ala Glu Lys Arg Glu Ser Gly Pr - #o Lys Gln Ala Ala Val         #         14705                                                               - Asn Ala Ala Val Gln Arg Val Gln Val Leu Pr - #o Asp Ala Asp Thr Leu         #     14850                                                                   - Leu His Phe Ala Thr Glu Ser Thr Pro Asp Gl - #y Phe Ser Cys Ser Ser         # 15005                                                                       - Ser Leu Ser Ala Leu Ser Leu Asp Glu Pro Ph - #e Ile Gln Lys Asp Val         #              15205  0                                                       - Glu Leu Arg Ile Met Pro Pro Val Gln Glu As - #n Asp Asn Gly Asn Glu         #             15350                                                           - Thr Glu Ser Glu Gln Pro Lys Glu Ser Asn Gl - #u Asn Gln Glu Lys Glu         #         15505                                                               - Ala Glu Lys Thr Ile Asp Ser Glu Lys Asp Le - #u Leu Asp Asp Ser Asp         #     15650                                                                   - Asp Asp Asp Ile Glu Ile Leu Glu Glu Cys Il - #e Ile Ser Ala Met Pro         # 15805                                                                       - Thr Lys Ser Ser Arg Lys Ala Lys Lys Pro Al - #a Gln Thr Ala Ser Lys         #              16005  0                                                       - Leu Pro Pro Pro Val Ala Arg Lys Pro Ser Gl - #n Leu Pro Val Tyr Lys         #             16150                                                           - Leu Leu Pro Ser Gln Asn Arg Leu Gln Pro Gl - #n Lys His Val Ser Phe         #         16305                                                               - Thr Pro Gly Asp Asp Met Pro Arg Val Tyr Cy - #s Val Glu Gly Thr Pro         #     16450                                                                   - Ile Asn Phe Ser Thr Ala Thr Ser Leu Ser As - #p Leu Thr Ile Glu Ser         # 16605                                                                       - Pro Pro Asn Glu Leu Ala Ala Gly Glu Gly Va - #l Arg Gly Gly Ala Gln         #              16805  0                                                       - Ser Gly Glu Phe Glu Lys Arg Asp Thr Ile Pr - #o Thr Glu Gly Arg Ser         #             16950                                                           - Thr Asp Glu Ala Gln Gly Gly Lys Thr Ser Se - #r Val Thr Ile Pro Glu         #         17105                                                               - Leu Asp Asp Asn Lys Ala Glu Glu Gly Asp Il - #e Leu Ala Glu Cys Ile         #     17250                                                                   - Asn Ser Ala Met Pro Lys Gly Lys Ser His Ly - #s Pro Phe Arg Val Lys         # 17405                                                                       - Lys Ile Met Asp Gln Val Gln Gln Ala Ser Al - #a Ser Ser Ser Ala Pro         #              17605  0                                                       - Asn Lys Asn Gln Leu Asp Gly Lys Lys Lys Ly - #s Pro Thr Ser Pro Val         #             17750                                                           - Lys Pro Ile Pro Gln Asn Thr Glu Tyr Arg Th - #r Arg Val Arg Lys Asn         #         17905                                                               - Ala Asp Ser Lys Asn Asn Leu Asn Ala Glu Ar - #g Val Phe Ser Asp Asn         #     18050                                                                   - Lys Asp Ser Lys Lys Gln Asn Leu Lys Asn As - #n Ser Lys Asp Phe Asn         # 18205                                                                       - Asp Lys Leu Pro Asn Asn Glu Asp Arg Val Ar - #g Gly Ser Phe Ala Phe         #              18405  0                                                       - Asp Ser Pro His His Tyr Thr Pro Ile Glu Gl - #y Thr Pro Tyr Cys Phe         #             18550                                                           - Ser Arg Asn Asp Ser Leu Ser Ser Leu Asp Ph - #e Asp Asp Asp Asp Val         #         18705                                                               - Asp Leu Ser Arg Glu Lys Ala Glu Leu Arg Ly - #s Ala Lys Glu Asn Lys         #     18850                                                                   - Glu Ser Glu Ala Lys Val Thr Ser His Thr Gl - #u Leu Thr Ser Asn Gln         # 19005                                                                       - Gln Ser Ala Asn Lys Thr Gln Ala Ile Ala Ly - #s Gln Pro Ile Asn Arg         #              19205  0                                                       - Gly Gln Pro Lys Pro Ile Leu Gln Lys Gln Se - #r Thr Phe Pro Gln Ser         #             19350                                                           - Ser Lys Asp Ile Pro Asp Arg Gly Ala Ala Th - #r Asp Glu Lys Leu Gln         #         19505                                                               - Asn Phe Ala Ile Glu Asn Thr Pro Val Cys Ph - #e Ser His Asn Ser Ser         #     19650                                                                   - Leu Ser Ser Leu Ser Asp Ile Asp Gln Glu As - #n Asn Asn Lys Glu Asn         # 19805                                                                       - Glu Pro Ile Lys Glu Thr Glu Pro Pro Asp Se - #r Gln Gly Glu Pro Ser         #              20005  0                                                       - Lys Pro Gln Ala Ser Gly Tyr Ala Pro Lys Se - #r Phe His Val Glu Asp         #             20150                                                           - Thr Pro Val Cys Phe Ser Arg Asn Ser Ser Le - #u Ser Ser Leu Ser Ile         #         20305                                                               - Asp Ser Glu Asp Asp Leu Leu Gln Glu Cys Il - #e Ser Ser Ala Met Pro         #     20450                                                                   - Lys Lys Lys Lys Pro Ser Arg Leu Lys Gly As - #p Asn Glu Lys His Ser         # 20605                                                                       - Pro Arg Asn Met Gly Gly Ile Leu Gly Glu As - #p Leu Thr Leu Asp Leu         #              20805  0                                                       - Lys Asp Ile Gln Arg Pro Asp Ser Glu His Gl - #y Leu Ser Pro Asp Ser         #             20950                                                           - Glu Asn Phe Asp Trp Lys Ala Ile Gln Glu Gl - #y Ala Asn Ser Ile Val         #         21105                                                               - Ser Ser Leu His Gln Ala Ala Ala Ala Ala Cy - #s Leu Ser Arg Gln Ala         #     21250                                                                   - Ser Ser Asp Ser Asp Ser Ile Leu Ser Leu Ly - #s Ser Gly Ile Ser Leu         # 21405                                                                       - Gly Ser Pro Phe His Leu Thr Pro Asp Gln Gl - #u Glu Lys Pro Phe Thr         #              21605  0                                                       - Ser Asn Lys Gly Pro Arg Ile Leu Lys Pro Gl - #y Glu Lys Ser Thr Leu         #             21750                                                           - Glu Thr Lys Lys Ile Glu Ser Glu Ser Lys Gl - #y Ile Lys Gly Gly Lys         #         21905                                                               - Lys Val Tyr Lys Ser Leu Ile Thr Gly Lys Va - #l Arg Ser Asn Ser Glu         #     22050                                                                   - Ile Ser Gly Gln Met Lys Gln Pro Leu Gln Al - #a Asn Met Pro Ser Ile         # 22205                                                                       - Ser Arg Gly Arg Thr Met Ile His Ile Pro Gl - #y Val Arg Asn Ser Ser         #              22405  0                                                       - Ser Ser Thr Ser Pro Val Ser Lys Lys Gly Pr - #o Pro Leu Lys Thr Pro         #             22550                                                           - Ala Ser Lys Ser Pro Ser Glu Gly Gln Thr Al - #a Thr Thr Ser Pro Arg         #         22705                                                               - Gly Ala Lys Pro Ser Val Lys Ser Glu Leu Se - #r Pro Val Ala Arg Gln         #     22850                                                                   - Thr Ser Gln Ile Gly Gly Ser Ser Lys Ala Pr - #o Ser Arg Ser Gly Ser         # 23005                                                                       - Arg Asp Ser Thr Pro Ser Arg Pro Ala Gln Gl - #n Pro Leu Ser Arg Pro         #              23205  0                                                       - Ile Gln Ser Pro Gly Arg Asn Ser Ile Ser Pr - #o Gly Arg Asn Gly Ile         #             23350                                                           - Ser Pro Pro Asn Lys Leu Ser Gln Leu Pro Ar - #g Thr Ser Ser Pro Ser         #         23505                                                               - Thr Ala Ser Thr Lys Ser Ser Gly Ser Gly Ly - #s Met Ser Tyr Thr Ser         #     23650                                                                   - Pro Gly Arg Gln Met Ser Gln Gln Asn Leu Th - #r Lys Gln Thr Gly Leu         # 23805                                                                       - Ser Lys Asn Ala Ser Ser Ile Pro Arg Ser Gl - #u Ser Ala Ser Lys Gly         #              24005  0                                                       - Leu Asn Gln Met Asn Asn Gly Asn Gly Ala As - #n Lys Lys Val Glu Leu         #             24150                                                           - Ser Arg Met Ser Ser Thr Lys Ser Ser Gly Se - #r Glu Ser Asp Arg Ser         #         24305                                                               - Glu Arg Pro Val Leu Val Arg Gln Ser Thr Ph - #e Ile Lys Glu Ala Pro         #     24450                                                                   - Ser Pro Thr Leu Arg Arg Lys Leu Glu Glu Se - #r Ala Ser Phe Glu Ser         # 24605                                                                       - Leu Ser Pro Ser Ser Arg Pro Ala Ser Pro Th - #r Arg Ser Gln Ala Gln         #              24805  0                                                       - Thr Pro Val Leu Ser Pro Ser Leu Pro Asp Me - #t Ser Leu Ser Thr His         #             24950                                                           - Ser Ser Val Gln Ala Gly Gly Trp Arg Lys Le - #u Pro Pro Asn Leu Ser         #         25105                                                               - Pro Thr Ile Glu Tyr Asn Asp Gly Arg Pro Al - #a Lys Arg His Asp Ile         #     25250                                                                   - Ala Arg Ser His Ser Glu Ser Pro Ser Arg Le - #u Pro Ile Asn Arg Ser         # 25405                                                                       - Gly Thr Trp Lys Arg Glu His Ser Lys His Se - #r Ser Ser Leu Pro Arg         #              25605  0                                                       - Val Ser Thr Trp Arg Arg Thr Gly Ser Ser Se - #r Ser Ile Leu Ser Ala         #             25750                                                           - Ser Ser Glu Ser Ser Glu Lys Ala Lys Ser Gl - #u Asp Glu Lys His Val         #         25905                                                               - Asn Ser Ile Ser Gly Thr Lys Gln Ser Lys Gl - #u Asn Gln Val Ser Ala         #     26050                                                                   - Lys Gly Thr Trp Arg Lys Ile Lys Glu Asn Gl - #u Phe Ser Pro Thr Asn         # 26205                                                                       - Ser Thr Ser Gln Thr Val Ser Ser Gly Ala Th - #r Asn Gly Ala Glu Ser         #              26405  0                                                       - Lys Thr Leu Ile Tyr Gln Met Ala Pro Ala Va - #l Ser Lys Thr Glu Asp         #             26550                                                           - Val Trp Val Arg Ile Glu Asp Cys Pro Ile As - #n Asn Pro Arg Ser Gly         #         26705                                                               - Arg Ser Pro Thr Gly Asn Thr Pro Pro Val Il - #e Asp Ser Val Ser Glu         #     26850                                                                   - Lys Ala Asn Pro Asn Ile Lys Asp Ser Lys As - #p Asn Gln Ala Lys Gln         # 27005                                                                       - Asn Val Gly Asn Gly Ser Val Pro Met Arg Th - #r Val Gly Leu Glu Asn         #              27205  0                                                       - Arg Leu Asn Ser Phe Ile Gln Val Asp Ala Pr - #o Asp Gln Lys Gly Thr         #             27350                                                           - Glu Ile Lys Pro Gly Gln Asn Asn Pro Val Pr - #o Val Ser Glu Thr Asn         #         27505                                                               - Glu Ser Ser Ile Val Glu Arg Thr Pro Phe Se - #r Ser Ser Ser Ser Ser         #     27650                                                                   - Lys His Ser Ser Pro Ser Gly Thr Val Ala Al - #a Arg Val Thr Pro Phe         # 27805                                                                       - Asn Tyr Asn Pro Ser Pro Arg Lys Ser Ser Al - #a Asp Ser Thr Ser Ala         #              28005  0                                                       - Arg Pro Ser Gln Ile Pro Thr Pro Val Asn As - #n Asn Thr Lys Lys Arg         #             28150                                                           - Asp Ser Lys Thr Asp Ser Thr Glu Ser Ser Gl - #y Thr Gln Ser Pro Lys         #         28305                                                               - Arg His Ser Gly Ser Tyr Leu Val Thr Ser Va - #l                             #      2840                                                                   - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3172 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: DP1(TB2)                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..630                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - GCA GTC GCC GCT CCA GTC TAT CCG GCA CTA GG - #A ACA GCC CCG GGN GGC           48                                                                          Ala Val Ala Ala Pro Val Tyr Pro Ala Leu Gl - #y Thr Ala Pro Gly Gly           #                 15                                                          - GAG ACG GTC CCC GCC ATG TCT GCG GCC ATG AG - #G GAG AGG TTC GAC CGG           96                                                                          Glu Thr Val Pro Ala Met Ser Ala Ala Met Ar - #g Glu Arg Phe Asp Arg           #             30                                                              - TTC CTG CAC GAG AAG AAC TGC ATG ACT GAC CT - #T CTG GCC AAG CTC GAG          144                                                                          Phe Leu His Glu Lys Asn Cys Met Thr Asp Le - #u Leu Ala Lys Leu Glu           #         45                                                                  - GCC AAA ACC GGC GTG AAC AGG AGC TTC ATC GC - #T CTT GGT GTC ATC GGA          192                                                                          Ala Lys Thr Gly Val Asn Arg Ser Phe Ile Al - #a Leu Gly Val Ile Gly           #     60                                                                      - CTG GTG GCC TTG TAC CTG GTG TTC GGT TAT GG - #A GCC TCT CTC CTC TGC          240                                                                          Leu Val Ala Leu Tyr Leu Val Phe Gly Tyr Gl - #y Ala Ser Leu Leu Cys           # 80                                                                          - AAC CTG ATA GGA TTT GGC TAC CCA GCC TAC AT - #C TCA ATT AAA GCT ATA          288                                                                          Asn Leu Ile Gly Phe Gly Tyr Pro Ala Tyr Il - #e Ser Ile Lys Ala Ile           #                 95                                                          - GAG AGT CCC AAC AAA GAA GAT GAT ACC CAG TG - #G CTG ACC TAC TGG GTA          336                                                                          Glu Ser Pro Asn Lys Glu Asp Asp Thr Gln Tr - #p Leu Thr Tyr Trp Val           #           110                                                               - GTG TAT GGT GTG TTC AGC ATT GCT GAA TTC TT - #C TCT GAT ATC TTC CTG          384                                                                          Val Tyr Gly Val Phe Ser Ile Ala Glu Phe Ph - #e Ser Asp Ile Phe Leu           #       125                                                                   - TCA TGG TTC CCC TTC TAC TAC ATG CTG AAG TG - #T GGC TTC CTG TTG TGG          432                                                                          Ser Trp Phe Pro Phe Tyr Tyr Met Leu Lys Cy - #s Gly Phe Leu Leu Trp           #   140                                                                       - TGC ATG GCC CCG AGC CCT TCT AAT GGG GCT GA - #A CTG CTC TAC AAG CGC          480                                                                          Cys Met Ala Pro Ser Pro Ser Asn Gly Ala Gl - #u Leu Leu Tyr Lys Arg           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - ATC ATC CGT CCT TTC TTC CTG AAG CAC GAG TC - #C CAG ATG GAC AGT GTG          528                                                                          Ile Ile Arg Pro Phe Phe Leu Lys His Glu Se - #r Gln Met Asp Ser Val           #               175                                                           - GTC AAG GAC CTT AAA GAC AAG TCC AAA GAG AC - #T GCA GAT GCC ATC ACT          576                                                                          Val Lys Asp Leu Lys Asp Lys Ser Lys Glu Th - #r Ala Asp Ala Ile Thr           #           190                                                               - AAA GAA GCG AAG AAA GCT ACC GTG AAT TTA CT - #G GGT GAA GAA AAG AAG          624                                                                          Lys Glu Ala Lys Lys Ala Thr Val Asn Leu Le - #u Gly Glu Glu Lys Lys           #       205                                                                   - AGC ACC TAAACCAGAC TAAACCAGAC TGGATGGAAA CTTCCTGCCC TC - #TCTGTACC           680                                                                          Ser Thr                                                                           210                                                                       - TTCCTACTGG AGCTTGATGT TATATTAGGG ACTGTGGTAT AATTATTTTA AT - #AATGTTGC        740                                                                          - CTTGGAAACA TTTTTGAGAT ATTAAAGATT GGAATGTGTT GTAAGTTTCT TT - #GCTTACTT        800                                                                          - TTACTGTCTA TATATATAGG GAGCACTTTA AACTTAATGC AGTGGGCAGT GT - #CCACGTTT        860                                                                          - TTGGAAAATG TATTTTGCCT CTGGGTAGGA AAAGATGTAT GTTGCTATCC TG - #CAGGAAAT        920                                                                          - ATAAACTTAA AATAAAATTA TATACCCCAC AGGCTGTGTA CTTTACTGGG CT - #CTCCCTGC        980                                                                          - ACGSATTTTC TCTGTAGTTA CATTTAGGRT AATCTTTATG GTTCTACTTC CT - #RTAATGTA       1040                                                                          - CAATTTTATA TAATTCNGRA ATGTTTTTAA TGTATTTGTG CACATGTACA TA - #TGGAAATG       1100                                                                          - TTACTGTCTG ACTACANCAT GCATCATGCT CATGGGGAGG GAGCAGGGGA AG - #GTTGTATG       1160                                                                          - TGTCATTTAT AACTTCTGTA CAGTAAGACC ACCTGCCAAA AGCTGGAGGA AC - #CATTGTGC       1220                                                                          - TGGTGTGGTC TACTAAATAA TACTTTAGGA AATACGTGAT TAATATGCAA GT - #GAACAAAG       1280                                                                          - TGAGAAATGA AATCGAATGG AGATTGGCCT GGTTGTTTCC GTAGTATATG GC - #ATATGAAT       1340                                                                          - ACCAGGATAG CTTTATAAAG CAGTTAGTTA GTTAGTTACT CACTCTAGTG AT - #AAATCGGG       1400                                                                          - AAATTTACAC ACACACACAC ACACACACAC ACACACACAC ACACACACAC AC - #ACACACAG       1460                                                                          - AGTACCCTGT AACTCTCAAT TCCCTGAAAA ACTAGTAATA CTGTCTTATC TG - #CTATAAAC       1520                                                                          - TTTACATATT TGTCTATTGT CAAGATGCTA CANTGGAMNC CATTTCTGGT TT - #TATCTTCA       1580                                                                          - NAGSGGAGAN ACATGTTGAT TTAGTCTTCT TTCCCAATCT TCTTTTTTAA MC - #CAGTTTNA       1640                                                                          - GGMNCTTCTG RAGATTTGYC CACCTCTGAT TACATGTATG TTCTYGTTTG TA - #TCATKAGC       1700                                                                          - AACAACATGC TAATGRCGAC ACCTAGCTCT RAGMGCAATT CTGGGAGANT GA - #RAGGNWGT       1760                                                                          - ATARAGTMNC CCATAATCTG CTTGGCAATA GTTAAGTCAA TCTATCTTCA GT - #TTTTCTCT       1820                                                                          - GGCCTTTAAG GTCAAACACA AGAGGCTTCC CTAGTTTACA AGTCAGAGTC AC - #TTGTAGTC       1880                                                                          - CATTTAAATG CCCTCATCCG TATTCTTTGT GTTGATAAGC TGCACAKGAC TA - #CATAGTAA       1940                                                                          - GTACAGANCA GTAAAGTTAA NNCGGATGTC TCCATTGATC TGCCAANTCG NT - #ATAGAGAG       2000                                                                          - CAATTTGTCT GGACTAGAAA ATCTGAGTTT TACACCATAC TGTTAAGAGT CC - #TTTTGAAT       2060                                                                          - TAAACTAGAC TAAAACAAGT GTATAACTAA ACTAACAAGA TTAAATATCC AG - #CCAGTACA       2120                                                                          - GTATTTTTTA AGGCAAATAA AGATGATTAG CTCACCTTGA GNTAACAATC AG - #GTAAGATC       2180                                                                          - ATNACAATGT CTCATGATGT NAANAATATT AAAGATATCA ATACTAAGTG AC - #AGTATCAC       2240                                                                          - NNCTAATATA ATATGGATCA GAGCATTTAT TTTGGGGAGG AAAACAGTGG TG - #ATTACCGG       2300                                                                          - CATTTTATTA AACTTAAAAC TTTGTAGAAA GCAAACAAAA TTGTTCTTGG GA - #GAAAATCA       2360                                                                          - ACTTTTAGAT TAAAAAAATT TTAAGTAWCT AGGAGTATTT AAATCCTTTT CC - #CATAAATA       2420                                                                          - AAAGTACAGT TTTCTTGGTG GCAGAATGAA AATCAGCAAC NTCTAGCATA TA - #GACTATAT       2480                                                                          - AATCAGATTG ACAGCATATA GAATATATTA TCAGACAAGA TGAGGAGGTA CA - #AAAGTTAC       2540                                                                          - TATTGCTCAT AATGACTTAC AGGCTAAAAN TAGNTNTAAA ATACTATATT AA - #ATTCTGAA       2600                                                                          - TGCAATTTTT TTTTGTTCCC TTGAGACCAA AATTTAAGTT AACTGTTGCT GG - #CAGTCTAA       2660                                                                          - GTGTAAATGT TAACAGCAGG AGAAGTTAAG AATTGAGCAG TTCTGTTGCA TG - #ATTTCCCA       2720                                                                          - AATGAAATAC TGCCTTGGCT AGAGTTTGAA AAACTAATTG AGCCTGTGCC TG - #GCTAGAAA       2780                                                                          - ACAAGCGTTT ATTTGAATGT GAATAGTGTT TCAAAGGTAT GTAGTTACAG AA - #TTCCTACC       2840                                                                          - AAACAGCTTA AATTCTTCAA GAAAGAATTC CTGCAGCAGT TATTCCCTTA CC - #TGAAGGCT       2900                                                                          - TCAATCATTT GGATCAACAA CTGCTACTCT CGGGAAGACT CCTCTACTCA CA - #GCTGAAGA       2960                                                                          - AAATGAGCAC ACCCTTCACA CTGTTATCAC CTATCCTGAA GATGTGATAC AC - #TGAATGGA       3020                                                                          - AATAAATAGA TGTAAATAAA ATTGAGWTCT CATTTAAAAA AAACCATGTG CC - #CAATGGGA       3080                                                                          - AAATGACCTC ATGTTGTGGT TTAAACAGCA ACTGCACCCA CTAGCACAGC CC - #ATTGAGCT       3140                                                                          #        3172      TCTG TCAGTGCCCC TC                                         - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 210 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Ala Val Ala Ala Pro Val Tyr Pro Ala Leu Gl - #y Thr Ala Pro Gly Gly         #                 15                                                          - Glu Thr Val Pro Ala Met Ser Ala Ala Met Ar - #g Glu Arg Phe Asp Arg         #             30                                                              - Phe Leu His Glu Lys Asn Cys Met Thr Asp Le - #u Leu Ala Lys Leu Glu         #         45                                                                  - Ala Lys Thr Gly Val Asn Arg Ser Phe Ile Al - #a Leu Gly Val Ile Gly         #     60                                                                      - Leu Val Ala Leu Tyr Leu Val Phe Gly Tyr Gl - #y Ala Ser Leu Leu Cys         # 80                                                                          - Asn Leu Ile Gly Phe Gly Tyr Pro Ala Tyr Il - #e Ser Ile Lys Ala Ile         #                 95                                                          - Glu Ser Pro Asn Lys Glu Asp Asp Thr Gln Tr - #p Leu Thr Tyr Trp Val         #           110                                                               - Val Tyr Gly Val Phe Ser Ile Ala Glu Phe Ph - #e Ser Asp Ile Phe Leu         #       125                                                                   - Ser Trp Phe Pro Phe Tyr Tyr Met Leu Lys Cy - #s Gly Phe Leu Leu Trp         #   140                                                                       - Cys Met Ala Pro Ser Pro Ser Asn Gly Ala Gl - #u Leu Leu Tyr Lys Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Ile Arg Pro Phe Phe Leu Lys His Glu Se - #r Gln Met Asp Ser Val         #               175                                                           - Val Lys Asp Leu Lys Asp Lys Ser Lys Glu Th - #r Ala Asp Ala Ile Thr         #           190                                                               - Lys Glu Ala Lys Lys Ala Thr Val Asn Leu Le - #u Gly Glu Glu Lys Lys         #       205                                                                   - Ser Thr                                                                         210                                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 434 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: TB1                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Val Ala Pro Val Val Val Gly Ser Gly Arg Al - #a Pro Arg His Pro Ala         #                15                                                           - Pro Ala Ala Met His Pro Arg Arg Pro Asp Gl - #y Phe Asp Gly Leu Gly         #            30                                                               - Tyr Arg Gly Gly Ala Arg Asp Glu Gln Gly Ph - #e Gly Gly Ala Phe Pro         #        45                                                                   - Ala Arg Ser Phe Ser Thr Gly Ser Asp Leu Gl - #y His Trp Val Thr Thr         #    60                                                                       - Pro Pro Asp Ile Pro Gly Ser Arg Asn Leu Hi - #s Trp Gly Glu Lys Ser         #80                                                                           - Pro Pro Tyr Gly Val Pro Thr Thr Ser Thr Pr - #o Tyr Glu Gly Pro Thr         #                95                                                           - Glu Glu Pro Phe Ser Ser Gly Gly Gly Gly Se - #r Val Gln Gly Gln Ser         #           110                                                               - Ser Glu Gln Leu Asn Arg Phe Ala Gly Phe Gl - #y Ile Gly Leu Ala Ser         #       125                                                                   - Leu Phe Thr Glu Asn Val Leu Ala His Pro Cy - #s Ile Val Leu Arg Arg         #   140                                                                       - Gln Cys Gln Val Asn Tyr His Ala Gln His Ty - #r His Leu Thr Pro Phe         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Val Ile Asn Ile Met Tyr Ser Phe Asn Ly - #s Thr Gln Gly Pro Arg         #               175                                                           - Ala Leu Trp Lys Gly Met Gly Ser Thr Phe Il - #e Val Gln Gly Val Thr         #           190                                                               - Leu Gly Ala Glu Gly Ile Ile Ser Glu Phe Th - #r Pro Leu Pro Arg Glu         #       205                                                                   - Val Leu His Lys Trp Ser Pro Lys Gln Ile Gl - #y Glu His Leu Leu Leu         #   220                                                                       - Lys Ser Leu Thr Tyr Val Val Ala Met Pro Ph - #e Tyr Ser Ala Ser Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Glu Thr Val Gln Ser Glu Ile Ile Arg As - #p Asn Thr Gly Ile Leu         #               255                                                           - Glu Cys Val Lys Glu Gly Ile Gly Arg Val Il - #e Gly Met Gly Val Pro         #           270                                                               - His Ser Lys Arg Leu Leu Pro Leu Leu Ser Le - #u Ile Phe Pro Thr Val         #       285                                                                   - Leu His Gly Val Leu His Tyr Ile Ile Ser Se - #r Val Ile Gln Lys Phe         #   300                                                                       - Val Leu Leu Ile Leu Lys Arg Lys Thr Tyr As - #n Ser His Leu Ala Glu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ser Thr Ser Pro Val Gln Ser Met Leu Asp Al - #a Tyr Phe Pro Glu Leu         #               335                                                           - Ile Ala Asn Phe Ala Ala Ser Leu Cys Ser As - #p Val Ile Leu Tyr Pro         #           350                                                               - Leu Glu Thr Val Leu His Arg Leu His Ile Gl - #n Gly Thr Arg Thr Ile         #       365                                                                   - Ile Asp Asn Thr Asp Leu Gly Tyr Glu Val Le - #u Pro Ile Asn Thr Gln         #   380                                                                       - Tyr Glu Gly Met Arg Asp Cys Ile Asn Thr Il - #e Arg Gln Glu Glu Gly         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Val Phe Gly Phe Tyr Lys Gly Phe Gly Ala Va - #l Ile Ile Gln Tyr Thr         #               415                                                           - Leu His Ala Ala Val Leu Gln Ile Thr Lys Il - #e Ile Tyr Ser Thr Leu         #           430                                                               - Leu Gln                                                                     - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 185 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: YS-39(TB2)                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Glu Leu Arg Arg Phe Asp Arg Phe Leu His Gl - #u Lys Asn Cys Met Thr         #                15                                                           - Asp Leu Leu Ala Lys Leu Glu Ala Lys Thr Gl - #y Val Asn Arg Ser Phe         #            30                                                               - Ile Ala Leu Gly Val Ile Gly Leu Val Ala Le - #u Tyr Leu Val Phe Gly         #        45                                                                   - Tyr Gly Ala Ser Leu Leu Cys Asn Leu Ile Gl - #y Phe Gly Tyr Pro Ala         #    60                                                                       - Tyr Ile Ser Ile Lys Ala Ile Glu Ser Pro As - #n Lys Glu Asp Asp Thr         #80                                                                           - Gln Trp Leu Thr Tyr Trp Val Val Tyr Gly Va - #l Phe Ser Ile Ala Glu         #                95                                                           - Phe Phe Ser Asp Ile Phe Leu Ser Trp Phe Pr - #o Phe Tyr Tyr Ile Leu         #           110                                                               - Lys Cys Gly Phe Leu Leu Trp Cys Met Ala Pr - #o Ser Pro Ser Asn Gly         #       125                                                                   - Ala Glu Leu Leu Tyr Lys Arg Ile Ile Arg Pr - #o Phe Phe Leu Lys His         #   140                                                                       - Glu Ser Gln Met Asp Ser Val Val Lys Asp Le - #u Lys Asp Lys Ala Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Thr Ala Asp Ala Ile Thr Lys Glu Ala Ly - #s Lys Ala Thr Val Asn         #               175                                                           - Leu Leu Gly Glu Glu Lys Lys Ser Thr                                         #           185                                                               - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 2843 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Met Ala Ala Ala Ser Tyr Asp Gln Leu Leu Ly - #s Gln Val Glu Ala Leu         #                15                                                           - Lys Met Glu Asn Ser Asn Leu Arg Gln Glu Le - #u Glu Asp Asn Ser Asn         #            30                                                               - His Leu Thr Lys Leu Glu Thr Glu Ala Ser As - #n Met Lys Glu Val Leu         #        45                                                                   - Lys Gln Leu Gln Gly Ser Ile Glu Asp Glu Al - #a Met Ala Ser Ser Gly         #    60                                                                       - Gln Ile Asp Leu Leu Glu Arg Leu Lys Glu Le - #u Asn Leu Asp Ser Ser         #80                                                                           - Asn Phe Pro Gly Val Lys Leu Arg Ser Lys Me - #t Ser Leu Arg Ser Tyr         #                95                                                           - Gly Ser Arg Glu Gly Ser Val Ser Ser Arg Se - #r Gly Glu Cys Ser Pro         #           110                                                               - Val Pro Met Gly Ser Phe Pro Arg Arg Gly Ph - #e Val Asn Gly Ser Arg         #       125                                                                   - Glu Ser Thr Gly Tyr Leu Glu Glu Leu Glu Ly - #s Glu Arg Ser Leu Leu         #   140                                                                       - Leu Ala Asp Leu Asp Lys Glu Glu Lys Glu Ly - #s Asp Trp Tyr Tyr Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Leu Gln Asn Leu Thr Lys Arg Ile Asp Se - #r Leu Pro Leu Thr Glu         #               175                                                           - Asn Phe Ser Leu Gln Thr Asp Met Thr Arg Ar - #g Gln Leu Glu Tyr Glu         #           190                                                               - Ala Arg Gln Ile Arg Val Ala Met Glu Glu Gl - #n Leu Gly Thr Cys Gln         #       205                                                                   - Asp Met Glu Lys Arg Ala Gln Arg Arg Ile Al - #a Arg Ile Gln Gln Ile         #   220                                                                       - Glu Lys Asp Ile Leu Arg Ile Arg Gln Leu Le - #u Gln Ser Gln Ala Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Glu Ala Glu Arg Ser Ser Gln Asn Lys His Gl - #u Thr Gly Ser His Asp         #               255                                                           - Ala Glu Arg Gln Asn Glu Gly Gln Gly Val Gl - #y Glu Ile Asn Met Ala         #           270                                                               - Thr Ser Gly Asn Gly Gln Gly Ser Thr Thr Ar - #g Met Asp His Glu Thr         #       285                                                                   - Ala Ser Val Leu Ser Ser Ser Ser Thr His Se - #r Ala Pro Arg Arg Leu         #   300                                                                       - Thr Ser His Leu Gly Thr Lys Val Glu Met Va - #l Tyr Ser Leu Leu Ser         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Met Leu Gly Thr His Asp Lys Asp Asp Met Se - #r Arg Thr Leu Leu Ala         #               335                                                           - Met Ser Ser Ser Gln Asp Ser Cys Ile Ser Me - #t Arg Gln Ser Gly Cys         #           350                                                               - Leu Pro Leu Leu Ile Gln Leu Leu His Gly As - #n Asp Lys Asp Ser Val         #       365                                                                   - Leu Leu Gly Asn Ser Arg Gly Ser Lys Glu Al - #a Arg Ala Arg Ala Ser         #   380                                                                       - Ala Ala Leu His Asn Ile Ile His Ser Gln Pr - #o Asp Asp Lys Arg Gly         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Arg Arg Glu Ile Arg Val Leu His Leu Leu Gl - #u Gln Ile Arg Ala Tyr         #               415                                                           - Cys Glu Thr Cys Trp Glu Trp Gln Glu Ala Hi - #s Glu Pro Gly Met Asp         #           430                                                               - Gln Asp Lys Asn Pro Met Pro Ala Pro Val Gl - #u His Gln Ile Cys Pro         #       445                                                                   - Ala Val Cys Val Leu Met Lys Leu Ser Phe As - #p Glu Glu His Arg His         #   460                                                                       - Ala Met Asn Glu Leu Gly Gly Leu Gln Ala Il - #e Ala Glu Leu Leu Gln         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Val Asp Cys Glu Met Tyr Gly Leu Thr Asn As - #p His Tyr Ser Ile Thr         #               495                                                           - Leu Arg Arg Tyr Ala Gly Met Ala Leu Thr As - #n Leu Thr Phe Gly Asp         #           510                                                               - Val Ala Asn Lys Ala Thr Leu Cys Ser Met Ly - #s Gly Cys Met Arg Ala         #       525                                                                   - Leu Val Ala Gln Leu Lys Ser Glu Ser Glu As - #p Leu Gln Gln Val Ile         #   540                                                                       - Ala Ser Val Leu Arg Asn Leu Ser Trp Arg Al - #a Asp Val Asn Ser Lys         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Lys Thr Leu Arg Glu Val Gly Ser Val Lys Al - #a Leu Met Glu Cys Ala         #               575                                                           - Leu Glu Val Lys Lys Glu Ser Thr Leu Lys Se - #r Val Leu Ser Ala Leu         #           590                                                               - Trp Asn Leu Ser Ala His Cys Thr Glu Asn Ly - #s Ala Asp Ile Cys Ala         #       605                                                                   - Val Asp Gly Ala Leu Ala Phe Leu Val Gly Th - #r Leu Thr Tyr Arg Ser         #   620                                                                       - Gln Thr Asn Thr Leu Ala Ile Ile Glu Ser Gl - #y Gly Gly Ile Leu Arg         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Asn Val Ser Ser Leu Ile Ala Thr Asn Glu As - #p His Arg Gln Ile Leu         #               655                                                           - Arg Glu Asn Asn Cys Leu Gln Thr Leu Leu Gl - #n His Leu Lys Ser His         #           670                                                               - Ser Leu Thr Ile Val Ser Asn Ala Cys Gly Th - #r Leu Trp Asn Leu Ser         #       685                                                                   - Ala Arg Asn Pro Lys Asp Gln Glu Ala Leu Tr - #p Asp Met Gly Ala Val         #   700                                                                       - Ser Met Leu Lys Asn Leu Ile His Ser Lys Hi - #s Lys Met Ile Ala Met         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Gly Ser Ala Ala Ala Leu Arg Asn Leu Met Al - #a Asn Arg Pro Ala Lys         #               735                                                           - Tyr Lys Asp Ala Asn Ile Met Ser Pro Gly Se - #r Ser Leu Pro Ser Leu         #           750                                                               - His Val Arg Lys Gln Lys Ala Leu Glu Ala Gl - #u Leu Asp Ala Gln His         #       765                                                                   - Leu Ser Glu Thr Phe Asp Asn Ile Asp Asn Le - #u Ser Pro Lys Ala Ser         #   780                                                                       - His Arg Ser Lys Gln Arg His Lys Gln Ser Le - #u Tyr Gly Asp Tyr Val         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Phe Asp Thr Asn Arg His Asp Asp Asn Arg Se - #r Asp Asn Phe Asn Thr         #               815                                                           - Gly Asn Met Thr Val Leu Ser Pro Tyr Leu As - #n Thr Thr Val Leu Pro         #           830                                                               - Ser Ser Ser Ser Ser Arg Gly Ser Leu Asp Se - #r Ser Arg Ser Glu Lys         #       845                                                                   - Asp Arg Ser Leu Glu Arg Glu Arg Gly Ile Gl - #y Leu Gly Asn Tyr His         #   860                                                                       - Pro Ala Thr Glu Asn Pro Gly Thr Ser Ser Ly - #s Arg Gly Leu Gln Ile         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Ser Thr Thr Ala Ala Gln Ile Ala Lys Val Me - #t Glu Glu Val Ser Ala         #               895                                                           - Ile His Thr Ser Gln Glu Asp Arg Ser Ser Gl - #y Ser Thr Thr Glu Leu         #           910                                                               - His Cys Val Thr Asp Glu Arg Asn Ala Leu Ar - #g Arg Ser Ser Ala Ala         #       925                                                                   - His Thr His Ser Asn Thr Tyr Asn Phe Thr Ly - #s Ser Glu Asn Ser Asn         #   940                                                                       - Arg Thr Cys Ser Met Pro Tyr Ala Lys Leu Gl - #u Tyr Lys Arg Ser Ser         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Asn Asp Ser Leu Asn Ser Val Ser Ser Ser As - #p Gly Tyr Gly Lys Arg         #               975                                                           - Gly Gln Met Lys Pro Ser Ile Glu Ser Tyr Se - #r Glu Asp Asp Glu Ser         #           990                                                               - Lys Phe Cys Ser Tyr Gly Gln Tyr Pro Ala As - #p Leu Ala His Lys Ile         #     10050                                                                   - His Ser Ala Asn His Met Asp Asp Asn Asp Gl - #y Glu Leu Asp Thr Pro         # 10205                                                                       - Ile Asn Tyr Ser Leu Lys Tyr Ser Asp Glu Gl - #n Leu Asn Ser Gly Arg         #              10405  0                                                       - Gln Ser Pro Ser Gln Asn Glu Arg Trp Ala Ar - #g Pro Lys His Ile Ile         #             10550                                                           - Glu Asp Glu Ile Lys Gln Ser Glu Gln Arg Gl - #n Ser Arg Asn Gln Ser         #         10705                                                               - Thr Thr Tyr Pro Val Tyr Thr Glu Ser Thr As - #p Asp Lys His Leu Lys         #     10850                                                                   - Phe Gln Pro His Phe Gly Gln Gln Glu Cys Va - #l Ser Pro Tyr Arg Ser         # 11005                                                                       - Arg Gly Ala Asn Gly Ser Glu Thr Asn Arg Va - #l Gly Ser Asn His Gly         #              11205  0                                                       - Ile Asn Gln Asn Val Ser Gln Ser Leu Cys Gl - #n Glu Asp Asp Tyr Glu         #             11350                                                           - Asp Asp Lys Pro Thr Asn Tyr Ser Glu Arg Ty - #r Ser Glu Glu Glu Gln         #         11505                                                               - His Glu Glu Glu Glu Arg Pro Thr Asn Tyr Se - #r Ile Lys Tyr Asn Glu         #     11650                                                                   - Glu Lys Arg His Val Asp Gln Pro Ile Asp Ty - #r Ser Leu Lys Tyr Ala         # 11805                                                                       - Thr Asp Ile Pro Ser Ser Gln Lys Gln Ser Ph - #e Ser Phe Ser Lys Ser         #              12005  0                                                       - Ser Ser Gly Gln Ser Ser Lys Thr Glu His Me - #t Ser Ser Ser Ser Glu         #             12150                                                           - Asn Thr Ser Thr Pro Ser Ser Asn Ala Lys Ar - #g Gln Asn Gln Leu His         #         12305                                                               - Pro Ser Ser Ala Gln Ser Arg Ser Gly Gln Pr - #o Gln Lys Ala Ala Thr         #     12450                                                                   - Cys Lys Val Ser Ser Ile Asn Gln Glu Thr Il - #e Gln Thr Tyr Cys Val         # 12605                                                                       - Glu Asp Thr Pro Ile Cys Phe Ser Arg Cys Se - #r Ser Leu Ser Ser Leu         #              12805  0                                                       - Ser Ser Ala Glu Asp Glu Ile Gly Cys Asn Gl - #n Thr Thr Gln Glu Ala         #             12950                                                           - Asp Ser Ala Asn Thr Leu Gln Ile Ala Glu Il - #e Lys Glu Lys Ile Gly         #         13105                                                               - Thr Arg Ser Ala Glu Asp Pro Val Ser Glu Va - #l Pro Ala Val Ser Gln         #     13250                                                                   - His Pro Arg Thr Lys Ser Ser Arg Leu Gln Gl - #y Ser Ser Leu Ser Ser         # 13405                                                                       - Glu Ser Ala Arg His Lys Ala Val Glu Phe Se - #r Ser Gly Ala Lys Ser         #              13605  0                                                       - Pro Ser Lys Ser Gly Ala Gln Thr Pro Lys Se - #r Pro Pro Glu His Tyr         #             13750                                                           - Val Gln Glu Thr Pro Leu Met Phe Ser Arg Cy - #s Thr Ser Val Ser Ser         #         13905                                                               - Leu Asp Ser Phe Glu Ser Arg Ser Ile Ala Se - #r Ser Val Gln Ser Glu         #     14050                                                                   - Pro Cys Ser Gly Met Val Ser Gly Ile Ile Se - #r Pro Ser Asp Leu Pro         # 14205                                                                       - Asp Ser Pro Gly Gln Thr Met Pro Pro Ser Ar - #g Ser Lys Thr Pro Pro         #              14405  0                                                       - Pro Pro Pro Gln Thr Ala Gln Thr Lys Arg Gl - #u Val Pro Lys Asn Lys         #             14550                                                           - Ala Pro Thr Ala Glu Lys Arg Glu Ser Gly Pr - #o Lys Gln Ala Ala Val         #         14705                                                               - Asn Ala Ala Val Gln Arg Val Gln Val Leu Pr - #o Asp Ala Asp Thr Leu         #     14850                                                                   - Leu His Phe Ala Thr Glu Ser Thr Pro Asp Gl - #y Phe Ser Cys Ser Ser         # 15005                                                                       - Ser Leu Ser Ala Leu Ser Leu Asp Glu Pro Ph - #e Ile Gln Lys Asp Val         #              15205  0                                                       - Glu Leu Arg Ile Met Pro Pro Val Gln Glu As - #n Asp Asn Gly Asn Glu         #             15350                                                           - Thr Glu Ser Glu Gln Pro Lys Glu Ser Asn Gl - #u Asn Gln Glu Lys Glu         #         15505                                                               - Ala Glu Lys Thr Ile Asp Ser Glu Lys Asp Le - #u Leu Asp Asp Ser Asp         #     15650                                                                   - Asp Asp Asp Ile Glu Ile Leu Glu Glu Cys Il - #e Ile Ser Ala Met Pro         # 15805                                                                       - Thr Lys Ser Ser Arg Lys Ala Lys Lys Pro Al - #a Gln Thr Ala Ser Lys         #              16005  0                                                       - Leu Pro Pro Pro Val Ala Arg Lys Pro Ser Gl - #n Leu Pro Val Tyr Lys         #             16150                                                           - Leu Leu Pro Ser Gln Asn Arg Leu Gln Pro Gl - #n Lys His Val Ser Phe         #         16305                                                               - Thr Pro Gly Asp Asp Met Pro Arg Val Tyr Cy - #s Val Glu Gly Thr Pro         #     16450                                                                   - Ile Asn Phe Ser Thr Ala Thr Ser Leu Ser As - #p Leu Thr Ile Glu Ser         # 16605                                                                       - Pro Pro Asn Glu Leu Ala Ala Gly Glu Gly Va - #l Arg Gly Gly Ala Gln         #              16805  0                                                       - Ser Gly Glu Phe Glu Lys Arg Asp Thr Ile Pr - #o Thr Glu Gly Arg Ser         #             16950                                                           - Thr Asp Glu Ala Gln Gly Gly Lys Thr Ser Se - #r Val Thr Ile Pro Glu         #         17105                                                               - Leu Asp Asp Asn Lys Ala Glu Glu Gly Asp Il - #e Leu Ala Glu Cys Ile         #     17250                                                                   - Asn Ser Ala Met Pro Lys Gly Lys Ser His Ly - #s Pro Phe Arg Val Lys         # 17405                                                                       - Lys Ile Met Asp Gln Val Gln Gln Ala Ser Al - #a Ser Ser Ser Ala Pro         #              17605  0                                                       - Asn Lys Asn Gln Leu Asp Gly Lys Lys Lys Ly - #s Pro Thr Ser Pro Val         #             17750                                                           - Lys Pro Ile Pro Gln Asn Thr Glu Tyr Arg Th - #r Arg Val Arg Lys Asn         #         17905                                                               - Ala Asp Ser Lys Asn Asn Leu Asn Ala Glu Ar - #g Val Phe Ser Asp Asn         #     18050                                                                   - Lys Asp Ser Lys Lys Gln Asn Leu Lys Asn As - #n Ser Lys Asp Phe Asn         # 18205                                                                       - Asp Lys Leu Pro Asn Asn Glu Asp Arg Val Ar - #g Gly Ser Phe Ala Phe         #              18405  0                                                       - Asp Ser Pro His His Tyr Thr Pro Ile Glu Gl - #y Thr Pro Tyr Cys Phe         #             18550                                                           - Ser Arg Asn Asp Ser Leu Ser Ser Leu Asp Ph - #e Asp Asp Asp Asp Val         #         18705                                                               - Asp Leu Ser Arg Glu Lys Ala Glu Leu Arg Ly - #s Ala Lys Glu Asn Lys         #     18850                                                                   - Glu Ser Glu Ala Lys Val Thr Ser His Thr Gl - #u Leu Thr Ser Asn Gln         # 19005                                                                       - Gln Ser Ala Asn Lys Thr Gln Ala Ile Ala Ly - #s Gln Pro Ile Asn Arg         #              19205  0                                                       - Gly Gln Pro Lys Pro Ile Leu Gln Lys Gln Se - #r Thr Phe Pro Gln Ser         #             19350                                                           - Ser Lys Asp Ile Pro Asp Arg Gly Ala Ala Th - #r Asp Glu Lys Leu Gln         #         19505                                                               - Asn Phe Ala Ile Glu Asn Thr Pro Val Cys Ph - #e Ser His Asn Ser Ser         #     19650                                                                   - Leu Ser Ser Leu Ser Asp Ile Asp Gln Glu As - #n Asn Asn Lys Glu Asn         # 19805                                                                       - Glu Pro Ile Lys Glu Thr Glu Pro Pro Asp Se - #r Gln Gly Glu Pro Ser         #              20005  0                                                       - Lys Pro Gln Ala Ser Gly Tyr Ala Pro Lys Se - #r Phe His Val Glu Asp         #             20150                                                           - Thr Pro Val Cys Phe Ser Arg Asn Ser Ser Le - #u Ser Ser Leu Ser Ile         #         20305                                                               - Asp Ser Glu Asp Asp Leu Leu Gln Glu Cys Il - #e Ser Ser Ala Met Pro         #     20450                                                                   - Lys Lys Lys Lys Pro Ser Arg Leu Lys Gly As - #p Asn Glu Lys His Ser         # 20605                                                                       - Pro Arg Asn Met Gly Gly Ile Leu Gly Glu As - #p Leu Thr Leu Asp Leu         #              20805  0                                                       - Lys Asp Ile Gln Arg Pro Asp Ser Glu His Gl - #y Leu Ser Pro Asp Ser         #             20950                                                           - Glu Asn Phe Asp Trp Lys Ala Ile Gln Glu Gl - #y Ala Asn Ser Ile Val         #         21105                                                               - Ser Ser Leu His Gln Ala Ala Ala Ala Ala Cy - #s Leu Ser Arg Gln Ala         #     21250                                                                   - Ser Ser Asp Ser Asp Ser Ile Leu Ser Leu Ly - #s Ser Gly Ile Ser Leu         # 21405                                                                       - Gly Ser Pro Phe His Leu Thr Pro Asp Gln Gl - #u Glu Lys Pro Phe Thr         #              21605  0                                                       - Ser Asn Lys Gly Pro Arg Ile Leu Lys Pro Gl - #y Glu Lys Ser Thr Leu         #             21750                                                           - Glu Thr Lys Lys Ile Glu Ser Glu Ser Lys Gl - #y Ile Lys Gly Gly Lys         #         21905                                                               - Lys Val Tyr Lys Ser Leu Ile Thr Gly Lys Va - #l Arg Ser Asn Ser Glu         #     22050                                                                   - Ile Ser Gly Gln Met Lys Gln Pro Leu Gln Al - #a Asn Met Pro Ser Ile         # 22205                                                                       - Ser Arg Gly Arg Thr Met Ile His Ile Pro Gl - #y Val Arg Asn Ser Ser         #              22405  0                                                       - Ser Ser Thr Ser Pro Val Ser Lys Lys Gly Pr - #o Pro Leu Lys Thr Pro         #             22550                                                           - Ala Ser Lys Ser Pro Ser Glu Gly Gln Thr Al - #a Thr Thr Ser Pro Arg         #         22705                                                               - Gly Ala Lys Pro Ser Val Lys Ser Glu Leu Se - #r Pro Val Ala Arg Gln         #     22850                                                                   - Thr Ser Gln Ile Gly Gly Ser Ser Lys Ala Pr - #o Ser Arg Ser Gly Ser         # 23005                                                                       - Arg Asp Ser Thr Pro Ser Arg Pro Ala Gln Gl - #n Pro Leu Ser Arg Pro         #              23205  0                                                       - Ile Gln Ser Pro Gly Arg Asn Ser Ile Ser Pr - #o Gly Arg Asn Gly Ile         #             23350                                                           - Ser Pro Pro Asn Lys Leu Ser Gln Leu Pro Ar - #g Thr Ser Ser Pro Ser         #         23505                                                               - Thr Ala Ser Thr Lys Ser Ser Gly Ser Gly Ly - #s Met Ser Tyr Thr Ser         #     23650                                                                   - Pro Gly Arg Gln Met Ser Gln Gln Asn Leu Th - #r Lys Gln Thr Gly Leu         # 23805                                                                       - Ser Lys Asn Ala Ser Ser Ile Pro Arg Ser Gl - #u Ser Ala Ser Lys Gly         #              24005  0                                                       - Leu Asn Gln Met Asn Asn Gly Asn Gly Ala As - #n Lys Lys Val Glu Leu         #             24150                                                           - Ser Arg Met Ser Ser Thr Lys Ser Ser Gly Se - #r Glu Ser Asp Arg Ser         #         24305                                                               - Glu Arg Pro Val Leu Val Arg Gln Ser Thr Ph - #e Ile Lys Glu Ala Pro         #     24450                                                                   - Ser Pro Thr Leu Arg Arg Lys Leu Glu Glu Se - #r Ala Ser Phe Glu Ser         # 24605                                                                       - Leu Ser Pro Ser Ser Arg Pro Ala Ser Pro Th - #r Arg Ser Gln Ala Gln         #              24805  0                                                       - Thr Pro Val Leu Ser Pro Ser Leu Pro Asp Me - #t Ser Leu Ser Thr His         #             24950                                                           - Ser Ser Val Gln Ala Gly Gly Trp Arg Lys Le - #u Pro Pro Asn Leu Ser         #         25105                                                               - Pro Thr Ile Glu Tyr Asn Asp Gly Arg Pro Al - #a Lys Arg His Asp Ile         #     25250                                                                   - Ala Arg Ser His Ser Glu Ser Pro Ser Arg Le - #u Pro Ile Asn Arg Ser         # 25405                                                                       - Gly Thr Trp Lys Arg Glu His Ser Lys His Se - #r Ser Ser Leu Pro Arg         #              25605  0                                                       - Val Ser Thr Trp Arg Arg Thr Gly Ser Ser Se - #r Ser Ile Leu Ser Ala         #             25750                                                           - Ser Ser Glu Ser Ser Glu Lys Ala Lys Ser Gl - #u Asp Glu Lys His Val         #         25905                                                               - Asn Ser Ile Ser Gly Thr Lys Gln Ser Lys Gl - #u Asn Gln Val Ser Ala         #     26050                                                                   - Lys Gly Thr Trp Arg Lys Ile Lys Glu Asn Gl - #u Phe Ser Pro Thr Asn         # 26205                                                                       - Ser Thr Ser Gln Thr Val Ser Ser Gly Ala Th - #r Asn Gly Ala Glu Ser         #              26405  0                                                       - Lys Thr Leu Ile Tyr Gln Met Ala Pro Ala Va - #l Ser Lys Thr Glu Asp         #             26550                                                           - Val Trp Val Arg Ile Glu Asp Cys Pro Ile As - #n Asn Pro Arg Ser Gly         #         26705                                                               - Arg Ser Pro Thr Gly Asn Thr Pro Pro Val Il - #e Asp Ser Val Ser Glu         #     26850                                                                   - Lys Ala Asn Pro Asn Ile Lys Asp Ser Lys As - #p Asn Gln Ala Lys Gln         # 27005                                                                       - Asn Val Gly Asn Gly Ser Val Pro Met Arg Th - #r Val Gly Leu Glu Asn         #              27205  0                                                       - Arg Leu Asn Ser Phe Ile Gln Val Asp Ala Pr - #o Asp Gln Lys Gly Thr         #             27350                                                           - Glu Ile Lys Pro Gly Gln Asn Asn Pro Val Pr - #o Val Ser Glu Thr Asn         #         27505                                                               - Glu Ser Ser Ile Val Glu Arg Thr Pro Phe Se - #r Ser Ser Ser Ser Ser         #     27650                                                                   - Lys His Ser Ser Pro Ser Gly Thr Val Ala Al - #a Arg Val Thr Pro Phe         # 27805                                                                       - Asn Tyr Asn Pro Ser Pro Arg Lys Ser Ser Al - #a Asp Ser Thr Ser Ala         #              28005  0                                                       - Arg Pro Ser Gln Ile Pro Thr Pro Val Asn As - #n Asn Thr Lys Lys Arg         #             28150                                                           - Asp Ser Lys Thr Asp Ser Thr Glu Ser Ser Gl - #y Thr Gln Ser Pro Lys         #         28305                                                               - Arg His Ser Gly Ser Tyr Leu Val Thr Ser Va - #l                             #      2840                                                                   - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 31 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: ral2(yeast)                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Leu Thr Gly Ala Lys Gly Leu Gln Leu Arg Al - #a Leu Arg Arg Ile Ala         #                15                                                           - Arg Ile Glu Gln Gly Gly Thr Ala Ile Ser Pr - #o Thr Ser Pro Leu             #            30                                                               - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 29 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: m3(mAChR)                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Leu Tyr Trp Arg Ile Tyr Lys Glu Thr Glu Ly - #s Arg Thr Lys Glu Leu         #                15                                                           - Ala Gly Leu Gln Ala Ser Gly Thr Glu Ala Gl - #u Thr Glu                     #            25                                                               - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 29 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -    (vii) IMMEDIATE SOURCE:                                                            (B) CLONE: MCC                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Leu Tyr Pro Asn Leu Ala Glu Glu Arg Ser Ar - #g Trp Glu Lys Glu Leu         #                15                                                           - Ala Gly Leu Arg Glu Glu Asn Glu Ser Leu Th - #r Ala Met                     #            25                                                               - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #    40            TTTT AATTGTAGTT TATCCATTTT                                 - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #    40            AATA TATTGTGTTC TTTTTAACAG                                 - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #    40            TGTT TTAAAATAAT TTTTTAAGCT                                 - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #    40            AAAA CTTGTTTCTA TTTTATTTAG                                 - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #    40            TAGT AAACATTGCC TTGTGTACTC                                 - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #    40            CCTT TTTTTAAAAA AAAAAAATAG                                 - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #    40            TACA ACTTATTTGA AACTTTAATA                                 - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #    40            CTTT TTTATTATTT GTGGTTTTAG                                 - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #    40            TAAG TGATAAAACA GYGAAGAGCT                                 - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #    40            ATTA GGTTTCTTGT TTTATTTTAG                                 - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #    40            TTTT GTTTGTGGGT ATAAAAATAG                                 - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #    40            CTGA TGTTAACTCC ATCTTAACAG                                 - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #    40            ATCA TATTTTTTAA AATTATTTAA                                 - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 64 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - CATGATGTTA TCTGTATTTA CCTATAGTCT AAATTATACC ATCTATAATG TG - #CTTAATTT         60                                                                          #             64                                                              - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 52 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - GTAACAGAAG ATTACAAACC CTGGTCACTA ATGCCATGAC TACTTTGCTA AG - #                 52                                                                          - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #                 46TTT TGTTTCTAAA CTCATTTGGC CCACAG                          - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                #    40            GTAC ATCGTAGTGC ATGTTTCAAA                                 - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 56 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - CATCATTGCT CTTCAAATAA CAAAGCATTA TGGTTTATGT TGATTTTATT TT - #TCAG             56                                                                          - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                # 43               TTTT AATGACATAG ACAATTACTG GTG                             - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                #    40            TTCC TCTTGCCCTT TTTAAATTAG                                 - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                # 44               TGTA TTTCTTAAGA TAGCTCAGGT ATGA                            - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 54 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                - GCTTGGCTTC AAGTTGNCTT TTTAATGATC CTCTATTCTG TATTTAATTT AC - #AG               54                                                                          - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 65 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                - GTACTATTTA GAATTTCACC TGTTTTTCTT TTTTCTCTTT TTCTTTGAGG CA - #GGGTCTCA         60                                                                          #            65                                                               - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 52 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - GCAACTAGTA TGATTTTATG TATAAATTAA TCTAAAATTG ATTAATTTCC AG - #                 52                                                                          - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 42 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #  42              TTAG TACTATAATA TGAATTTCAT GT                              - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                #    40            GACC CATATTCAGA AACTTACTAG                                 - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 54 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - GTATATATAG AGTTTTATAT TACTTTTAAA GTACAGAATT CATACTCTCA AA - #AA               54                                                                          - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                #   41             TGTG ATCTCTTGAT TTTTATTTCA G                               - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                #  18              TC                                                         - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                #  18              TG                                                         - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                # 20               CTGC                                                       - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                # 19               GGA                                                        - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                #                24TGAT ATAC                                                  - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                #                23TATA CAG                                                   - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                #21                TTTT C                                                     - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                # 20               CTGA                                                       - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                #                 22TGC AA                                                    - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #                 22AGG TA                                                    - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                # 19               TTG                                                        - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                # 20               GGAC                                                       - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                #21                ACTG C                                                     - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                # 20               CCTC                                                       - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                #                24TATT TAGT                                                  - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                #                 22GAA CC                                                    - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                #                24TTCT TTTG                                                  - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                #                23TGTT GTG                                                   - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                #                24AGCC TAAC                                                  - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                #                 22TAC CA                                                    - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                # 20               AAGG                                                       - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                #             27   ACAA TTAAAAG                                               - (2) INFORMATION FOR SEQ ID NO:61:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                #                24CTTG AAGT                                                  - (2) INFORMATION FOR SEQ ID NO:62:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                #                23ATTT GAG                                                   - (2) INFORMATION FOR SEQ ID NO:63:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                #                24AATT TCCC                                                  - (2) INFORMATION FOR SEQ ID NO:64:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                #                23CACA AGG                                                   - (2) INFORMATION FOR SEQ ID NO:65:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                #                23TGCT GAT                                                   - (2) INFORMATION FOR SEQ ID NO:66:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                #                24ACCT AGGT                                                  - (2) INFORMATION FOR SEQ ID NO:67:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                #               25 TGAT TAACG                                                 - (2) INFORMATION FOR SEQ ID NO:68:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                #             27   CCTA ATAGCTC                                               - (2) INFORMATION FOR SEQ ID NO:69:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                #                24TTAT TTCT                                                  - (2) INFORMATION FOR SEQ ID NO:70:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                #                24CCAC AAAC                                                  - (2) INFORMATION FOR SEQ ID NO:71:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                #                23TTTT TGC                                                   - (2) INFORMATION FOR SEQ ID NO:72:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                #                23ATCT TGC                                                   - (2) INFORMATION FOR SEQ ID NO:73:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                #                24ATAC CATC                                                  - (2) INFORMATION FOR SEQ ID NO:74:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                # 20               CCAG                                                       - (2) INFORMATION FOR SEQ ID NO:75:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                #                24CTAA ACTC                                                  - (2) INFORMATION FOR SEQ ID NO:76:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                #21                CACG C                                                     - (2) INFORMATION FOR SEQ ID NO:77:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                #                23TGAT GAC                                                   - (2) INFORMATION FOR SEQ ID NO:78:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                #                 22ACG AT                                                    - (2) INFORMATION FOR SEQ ID NO:79:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                #                24CAAA TAAC                                                  - (2) INFORMATION FOR SEQ ID NO:80:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                #                24TCCA CCAG                                                  - (2) INFORMATION FOR SEQ ID NO:81:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                #                23CTCT TGC                                                   - (2) INFORMATION FOR SEQ ID NO:82:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                #                24AATA CATG                                                  - (2) INFORMATION FOR SEQ ID NO:83:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                #               25 ATTC TGTAT                                                 - (2) INFORMATION FOR SEQ ID NO:84:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                #                24CCTC AAAG                                                  - (2) INFORMATION FOR SEQ ID NO:85:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                #                23TAGC ATT                                                   - (2) INFORMATION FOR SEQ ID NO:86:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                #                 22TAG GA                                                    - (2) INFORMATION FOR SEQ ID NO:87:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                #                 22GTT TC                                                    - (2) INFORMATION FOR SEQ ID NO:88:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                #                 22GAG TA                                                    - (2) INFORMATION FOR SEQ ID NO:89:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                #                 22GTG AC                                                    - (2) INFORMATION FOR SEQ ID NO:90:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                #                23CATG AAG                                                   - (2) INFORMATION FOR SEQ ID NO:91:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                #21                CTCC C                                                     - (2) INFORMATION FOR SEQ ID NO:92:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                #21                GTAC G                                                     - (2) INFORMATION FOR SEQ ID NO:93:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                #                 22GTC CC                                                    - (2) INFORMATION FOR SEQ ID NO:94:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                #                24TCTT CCTC                                                  - (2) INFORMATION FOR SEQ ID NO: 95:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                #               25 CAGT GTGGA                                                 - (2) INFORMATION FOR SEQ ID NO: 96:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                #                24TGAG TTTG                                                  - (2) INFORMATION FOR SEQ ID NO: 97:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                #  18              AG                                                         - (2) INFORMATION FOR SEQ ID NO: 98:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                # 19               CAA                                                        - (2) INFORMATION FOR SEQ ID NO: 99:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                #21                AACA A                                                     - (2) INFORMATION FOR SEQ ID NO: 100:                                         -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                               # 19               CAG                                                        - (2) INFORMATION FOR SEQ ID NO: 101:                                         -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                               #  18              AG                                                         - (2) INFORMATION FOR SEQ ID NO: 102:                                         -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Homo sapi - #ens                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                               #  18              GA                                                         __________________________________________________________________________

                                      TABLE I                                     __________________________________________________________________________    APC EXONS                                                                     EXON     EXON                                                                 NUCLEOTIDES.sup.1                                                                      BOUNDARY SEQUENCE.sup.2                                              __________________________________________________________________________    822 to 930                                                                             ctgatgttatcgtatttacctatagtctaaattataccatctataatgtgcttaatttttag/GG             TTCA . . .                                                                    . . . ACCAAG/gtaacagaagattacaaaccctggtcactaatgccatgactactttgctaag     931 to 1309                                                                           ggatattaaagtcgtaattttgtttctaaactcatttggcccacag/GTGGAA . . .                   . . . ATCCAA/gtatgttctctatagtgtacatcgtagtgcatg                       1310 to 1405                                                                           catcattgctcttcaaataacaaagcattatggtttatgttgattttatttttcag/TGCCAG               . . .                                                                         . . . AACTAG/gtaagacaaaaatgtttttaatgacatagacaattactggtg              1406 to 1545                                                                           tagatgattgtctttttcctcttgccctttttaaattag/GGGGAC . . .                          . . . AACAAG/gtatgttttataacatgtatttcttaagatagctcggtatga              1546 to 1623                                                                           gcttggcttcaagttgtctttttaatgatcctctattctgtatttaatttacag/GCTACG .               . .                                                                           . . . CAGCAG/gtactatttagaatttcacctgtttttctttttctctttttctttgaggcag             ggtctcactctg                                                         1624 to 1740                                                                           gcaactagtatgattttatgtataaataattctaaaattgattaatttgcag/GTTATT . .               .                                                                             . . . AAAAAG/gtacctttgaaaacatttgtactataatatgaatttcatgt               1741 to 1955                                                                           caactctaattagatgacccatattcagaaacttactag/GAATCA . . .                          . . . CCACAG/gtatatatagagttttatattacttttaagtacagaattcatactctcaaaa    1956 to 8973                                                                           tcttgatttttatttcag/GCAAAT . . .                                               . . . GGTATTTATGCAAAAAAAAATGTTTTTGT                                  __________________________________________________________________________     .sup.1 Relative to predicted translation initiation site                      .sup.2 Small case letters represent introns, large case letters represent     exons                                                                         .sup.3 The entire 3' end of the cloned APC cDNA (nt 19568973) appeared to     be encoded in this exon, as indicated by restriction endonuclease mapping     and sequencing of cloned genomic DNA. The ORF ended at at 8535. The           extreme 3' end of the APC transcript has not yet been indentified.            .sup.4 The first line of sequence is (SEQ ID NO: 24); the second line of      sequence is (SEQ ID NO: 25); the third line of sequence is (SEQ ID NO:        26); the fourth line of sequence is (SEQ ID NO: 27); the fifth line of        sequence is (SEQ ID NO: 28); the sixth line of sequence is (SEQ ID NO:        29); the seventh line of sequence is (SEQ ID NO: 30); the eighth line of      sequence is (SEQ ID NO: 31); the ninth line of sequence is (SEQ ID NO:        32); the tenth line of sequence is (SEQ ID NO: 33); the eleventh line of      sequence is (SEQ ID NO: 34); the twelfth line of sequence is (SEQ ID NO:      35); the thirteenth line of sequence is (SEQ ID NO: 36); the fourteenth       line of sequence is (SEQ ID NO: 37); the fifteenth line of sequence is        (SEQ ID NO: 38); and the sixteenth line of sequence is (SEQ ID NO: 1).   

                  TABLE IIA                                                       ______________________________________                                        Germline mutations of the APC gene in FAP and GS Patients                                              Amino        Extra-                                  Pa-  Co-    Nucleotide   Acid         colonic                                 tient                                                                              don    Change       Change  Age  Disease                                 ______________________________________                                         93  279    TCA → TGA                                                                           Ser → Stop                                                                     39   Mindibular                                                                    Osteoma                                  24  301    CGA → TGA                                                                           Arg → Stop                                                                     46   None                                     34  301    CGA → TGA                                                                           Arg → Stop                                                                     27   Desmoid                                                                       Tumor                                    21  413    CGC → TGC                                                                           Arg → Cys                                                                      24   Mandibular                                                                    Osteoma                                  60  712    TCA → TGA                                                                           Ser → Stop                                                                     37   Mandibular                                                                    Osteoma                                 3746 243    CAGAG → CAG                                                                         splice-                                                                       junction                                             3460 301    CGA → TGA                                                                           Arg → stop                                    3827 456    CTTTCA → CTT                                                                        frameshift                                                       CA                                                                3712 500    T → G Tyr → Stop                                    ______________________________________                                         *The mutated nucleotides are underlined.                                 

                  TABLE IIB                                                       ______________________________________                                        Somatic Mutations in Sporadic CRC Patients                                                                       AMINO                                      PA-                                ACID                                       TIENT CODON.sup.1                                                                              NUCLEOTIDE CHANGE CHANGE                                     ______________________________________                                        T35   MCC 12     GAG/gtaaga →                                                                             (Splice                                          GAG/gtaaaa Donor)                                                       T16   MCC 145    vtcag/GGA →                                                                              (Splice                                                     atcag/GGA         Acceptor)                                  T47   MCC 267    CGG → CTG  Arg → Leu                           T81   MCC 490    TCG → TTG  Ser → Leu                           T35   MCC 506    CGG → CAG  Arg → Gln                           T91   MCC 698    GCT ∝3 GTT Ala → Val                           T34   APC 288    CCAGT → CCCAGCCAGT                                                                       (Insertion)                                T27   APC 331    CGA → TGA  Arg → Stop                          T135  APC 437    CAA/gtaa → CAA/gcaa                                                                      (Splice                                                                       Donor)                                     T201  APC 1338   CAG → TAG  Gln → Stop                          ______________________________________                                         For splice site mutations, the codon nearest to the mutation is listed        The underlined nucleotides were mutant; small case letters represent          introns, large case letters represent exons                              

                                      TABLE III                                   __________________________________________________________________________    Sequences of Primers Used for SSCP Analyses                                   Exon                                                                             Primer 1            Primer 2                                               __________________________________________________________________________    DP1                                                                           UP-TCCCCGCCTGCCGCTCTC  RP-GCAGCGGCGGCTCCCGTG                                  UP-GTGAACGGCTCTCATGCTGC                                                                              RP-ACGTGCGGGGAGGAATGGA                                 UP-ATGATATCTTACCAAATGATATAC                                                                          RP-TTATTCCTACTTCTTCTATACAG                             UP-TACCCATGCTGGCTCTTTTTC                                                                             RP-TGGGGCCATCTTGTTCCTGA                                UP-ACATTAGGCACAAAGCTTGCAA                                                                            RP-ATCAAGCTCCAGTAAGAAGGTA                              SRP19                                                                         UP-TGCGGCTCCTGGGTTGTTG RP-GCCCCTTCCTTTCTGAGGAC                                UP-TTTTCTCCTGCCTCTTACTGC                                                                             RP-ATGACACCCCCCATTCCCTC                                UP-CCACTTAAAGCACATATATTTAGT                                                                          RP-GTATGGAAAATAGTGAAGAACC                              UP-TTCTTAAGTCCTGTTTTTCTTTTG                                                                          RP-TTTAGAACCTTTTTTGTGTTGTG                             UP-CTCAGATTATACACTAAGCCTAAC                                                                          RP-CATGTCTCTTACAGTAGTACCA                              DP2.5                                                                            UP-AGGTCCAAGGGTAGCCAAGG*                                                                          RP-TAAAAATGGATAAACTACAATTAAAAG                            UP-AAATACAGAATCATGTCTTGAAGT                                                                       RP-ACACCTAAAGATGACAATTTGAG                                UP-TAACTTAGATAGCAGTAATTTCCC*                                                                      RP-ACAATAAACTGGAGTACACAAGG                                UP-ATAGGTCATTGCTTCTTGCTGAT*                                                                       RP-TGAATTTTAATGGATTACCTAGGT                               UP-CTTTTTTTGCTTTTACTGATTAACG                                                                      RP-TGTAATTCATTTTATTCTAATACCTC                             UP-GGTAGCCATAGTAGATTATTTCT                                                                        RP-CTACCTATTTTTATACCCACAAAC                               UP-AAGAAAGCCTACACCATTTTTGC                                                                        RP-GATCATTCTTAGAACCATCTTGC                                UP-ACCTTAGTCTAAATTATACCATC                                                                        RP-GTCATGGCATTACTGACCAG                                   UP-AGTCGTAATTTTGTTTCTAAACTC                                                                       RP-TGAAGGACTCCGATTTCACCC*                                 UP-TCATTCACTCACAGCTGATGAC*                                                                        RP-GCTTTGAAACATGCACTACGAT                                 UP-AAACATCATTGCTCTTCAAATAAC                                                                       RP-TACCATGATTTAAAAATCCACCAG                               UP-GATGATTGTCTTTTTCCTCTTGC                                                                        RP-CTGAGCTATCTTAAGAAATACATG                               UP-TTTTAAATGATCCTCTATTCTGTAT                                                                      RP-ACAGAGTCAGACCCTCCCTCAAAG                               UP-TTTCTATTCTTACTGCTAGCATT                                                                        RP-ATACACAGGTAAGAAATTAGGA                                 UP-TAGATGACCCATATTCTCTTTC                                                                         RP-CAATTAGGTCTTTTTGAGAGTA                              3-A                                                                              UP-GTTACTGCATACACATTGTGAC                                                                         RP-GCTTTTTGTTTCGTAACATGAAG*                            B  UP-AGTACAAGGATGCCAATATTATG*                                                                       RP-ACTTCTATCTTTTTCAGAACGAG*                            C  UP-ATTGAATACTACAGTGTTACCC*                                                                        RP-CTTGTATTCTAATTTGGCATAAGG*                           D  UP-CTGCCCATACACATTCAAACAC*                                                                        RP-TGTTTGCGTCTTGCCCATCTT*                              E  UP-AGTCTTAAATATTCAGATGAGCAG*                                                                      RP-GTTTCTCTTCATTATATTTTATGCTA*                         F  UP-AAGCCTACCAATTATAGTGAACG*                                                                       RP-AGCTGATGACAAAGATGATAATC*                            G  UP-AAGAAACAATACAGACTTATTGTG*                                                                      RP-ATGAGTGGGGTCTCCTGAAC*                               H  UPATCTCCCTCCAAAAGTGGTGC*                                                                          RP-TCCATCTGGAGTACTTTCTGTG*                             I  UP-AGTAAATGCTGCAGTTCAGAGG*                                                                        RP-CCGTGGCATATCATCCCCC*                                J  UP-CCCAGACTGCTTCAAAATTACC*                                                                        RP-GAGCCTCATCTGTACTTCTGC*                              K  UP-CCCTCCAAATGAGTTAGCTGC*                                                                         RP-TTGTGGTATAGGTTTTACTGGTG*                            L  UP-ACCCAACAAAAATCAGTTAGATG*                                                                       RP-GTGGCTGGTAACTTTAGCCTC*                              N  UP-ATGATGTTGACCTTTCCAGGG*                                                                         RP-ATTGTGTAACTTTTCATCAGTTGC*                           O  UP-AAGATGACCTGTTGCAGGAATG*                                                                        RP-GAATCAGACCAAGCTTGTCTAGAT*                           P  UP-CAATAGTAAGTAGTTTACATCAAG*                                                                      RP-AAACAGGACTTGTACTGTAGGA*                             Q  UP-CAGCCCCTTCAAGCAAACATC*                                                                         RP-GAGGACTTATTCCATTTCTACC*                             R  UP-CAGTCTCCTGGCCGAACTC*                                                                           RP-GTTGACTGGCGTACTAATACAG*                             S  UP-TGGTAATGGAGCCAATAAAAAGG*                                                                       RP-TGGGACTTTTCGCCATCCAC*                               T  UP-TGTCTCTATCCACACATTCGTC*                                                                        RP-ATGTTTTTCATCCTCACTTTTTGC*                           U  UP-GGAGAAGAACTGGAAGTTCATC*                                                                        RP-TTGAATCTTTAATGTTTGGATTTGC*                          V  UP-TCTCCCACAGGTAATACTCCC                                                                          RP-GCTACAACTGAATGGGGTACG                               W  UP-CAGGACAAAATAATCCTGTCCC                                                                         RP-ATTTTCTTACTTTCATTCTTCCTC                            __________________________________________________________________________     All primers are read in the 5' to 3' direction. the first primer in each      pair lies 5' of the exon ir amplifies: the second primer lies 3' of the       exon it amplifies. Primers that lie within the exon are identified by an      asterisk. UP represnets the  2ImI3 universal primer sequence: RP              represnets the MI3 reverse primer sequence.                                   Primer 1 of DP1 exons 1, 2, 3, 4, and 5 are shown in SEQ ID NOS: 39, 41,      43, 45, and 47, respectively. Primer 2 of DP1 exons 1, 2, 3, 4, and 5 are     shown in SEQ ID NOS: 40, 42, 44, 46, and 48, respectively. Primer 1 of        SRP19 exons 1, 2, 3, 4, and 5 are shown in SEQ ID NOS: 49, 51, 53, 55, an     57, respectively. Primer 2 of SRP19 exons 1, 2, 3, 4, and 5 are shown in      SEQ ID NOS: 50, 52, 54, 56, and 58, respectively. Primer 1 of DP2.5 exons     1, 2, 3, 4, 5, 6, 7, 8, 9, 9a, 10, 11, 12, 13, 14, and 15A are shown in       SEQ ID NOS: 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,       and 89, respectively. Primer 2 of DP2.5 exons 1, 2, 3, 4, 5, 6, 7, 8, 9,      9a, 10, 11, 12, 13, 14, and 15A are shown in SEQ ID NOS: 60, 62, 64, 66,      68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90, respectively. Primer      and primer 2 of DP2.5 exon 15B, C, D, E, F, G, H, I, J, K, L, M, N, O, P,     Q, R, S, T, and U are shown in SEQ ID NO: 1.                             

                                      TABLE IV                                    __________________________________________________________________________    Seven Different Versions of the 20-Amino Acid Repeat                          Consensus:                                                                             F . V E . T P . C F S R . S S L S S L S                              __________________________________________________________________________    1262:    Y C V E D T P I C F S R C S S L S S L S                              1376:    H Y V Q E T P L M F S R C T S V S S    L                                                                        D                                  1492:    F A T E S T P D G F S C S S S L S A    L                                                                        S                                  1643:    Y C V E G T P I N F S T A T S L S D    L                                                                        T                                  1848:    T P I E G T P Y C F S R N D S L S S    L                                                                        D                                  1953:    F A I E N T P V C P S H N S S L S S    L                                                                        S                                  2013:    F H V E D T P V C F S R N S S L S S    L                                                                        S                                  __________________________________________________________________________     Numbers denote the first amino acid of each repeat. The consensus sequenc     at the top reflects a majority amino acid at a given position.           

We claim:
 1. A cDNA molecule having the nucleotide sequence shown in SEQID NO: 1 or its complement.
 2. An isolated DNA molecule having thenucleotide sequence shown in SEQ ID NO:1 or its complement.
 3. A cDNAmolecule which encodes a protein having the amino acid sequence shown in.[.FIG. 3 or 7 (.].SEQ ID NO: 7 or 2.[.).]..
 4. An isolated DNA moleculewhich encodes a protein having the amino acid sequence shown in .[.FIG.3 or 7 (.].SEQ ID NO: 7 or 2.[.).]..
 5. A nucleic acid probecomplementary to all or part of human wild-type APC gene codingsequences or the complement of said sequences such that said probeselectively hybridizes under stringent conditions to said APC gene oridentifies endogenous, random modifications in said APC gene.
 6. Thenucleic acid probe of claim 5 which hybridizes to all or part of an exonselected from the group consisting of: (1) nucleotides 822 to 930; (2)nucleotides 931 to to 1309; (3) nucleotides 1406 to 1545; and (4)nucleotides 1956 to 2256 .Iadd.as shown in SEQ ID NO: 1.Iaddend..
 7. Aset of probes useful for detecting alteration of wild-type APC genescomprising a plurality of nucleic acid probes wherein said set iscomplementary to all nucleotides of the APC gene coding sequences asshown in SEQ ID NO:1 or the complement of said sequences.
 8. A pair ofsingle stranded DNA primers for determination of a nucleotide sequenceof an APC gene by polymerase chain reaction, the sequence of saidprimers being derived from said APC gene, wherein the use of saidprimers in a polymerase chain reaction results in synthesis of DNAhaving all or part of the sequence shown in .[.FIG. 7 (.].SEQ IDNO:1.[.).]..
 9. The pair of primers of claim 8 which have restrictionenzyme sites at each 5' end.
 10. The pair of primers of claim 8 havingsequences complementary to all or part of one or more APC introns.